Team:UC-Santa Cruz/Notebook2

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<p class=MsoNormal><span style='font-size:19.0pt;font-family:Arial;mso-bidi-font-family:
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CB15N (for immediate use)</span></b><span style='font-size:10.0pt;font-family:
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style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span
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<p class=MsoNormal style='text-indent:.5in'><span style='font-size:15.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>1. Check
 +
OD600 of culture</span><span style='font-size:10.0pt;font-family:Times;
 +
mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='text-indent:.5in'><span style='font-size:15.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>2.
 +
Balance culture tubes &lt; 0.01 g difference</span><span style='font-size:10.0pt;
 +
font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='text-indent:.5in'><span style='font-size:15.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>3. Spin
 +
at 3000 rpm 4Cº for 15 minutes</span><span style='font-size:10.0pt;font-family:
 +
Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='text-indent:.5in'><span style='font-size:15.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>4.
 +
Discard supernatant</span><span style='font-size:10.0pt;font-family:Times;
 +
mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='text-indent:.5in'><span style='font-size:15.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>5. Add 10
 +
mL Milli Q water</span><span style='font-size:10.0pt;font-family:Times;
 +
mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='text-indent:.5in'><span style='font-size:15.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>6. Spin
 +
at 3000 rpm 4Cº for 15 minutes</span><span style='font-size:10.0pt;font-family:
 +
Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='text-indent:.5in'><span style='font-size:15.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>7.
 +
Discard supernatant.</span><span style='font-size:10.0pt;font-family:Times;
 +
mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='text-indent:.5in'><span style='font-size:15.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>8. Repeat
 +
step 5-7 three times</span><span style='font-size:10.0pt;font-family:Times;
 +
mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;9.
 +
Make aliquots and put in deep freezer.</span><span style='font-size:10.0pt;
 +
font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br
 +
style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><b><span style='font-size:15.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>2. Miniprep on <i>E. coli</i>
 +
cultures</span></b><span style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:
 +
"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:15.0pt;mso-bidi-font-size:12.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'><span
 +
style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span
 +
style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>1.Spin <i>E. coli</i> culture at 3000rpm for 5 minutes</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:15.0pt;mso-bidi-font-size:12.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'><span
 +
style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span
 +
style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>2. Discard supernatant.</span><span style='font-size:10.0pt;
 +
font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:15.0pt;mso-bidi-font-size:12.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'><span
 +
style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span
 +
style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>3. Resuspend in 6mL of Milli Q water.</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:15.0pt;mso-bidi-font-size:12.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'><span
 +
style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span
 +
style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>4. Add 1mL 6x Lysis buffer, invert 4-6 times, let sit for 5 minutes</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='text-align:justify'><span style='font-size:15.0pt;
 +
mso-bidi-font-size:12.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'><span style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span
 +
style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>5. Add entire mix to blue zymo-midi-filter and &nbsp;column, place
 +
&nbsp;in 50mL conical tube. spin 5000rcf for 6 minutes</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:15.0pt;mso-bidi-font-size:12.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'><span
 +
style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span
 +
style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>6.Remove and discard filter and place column on collection tube.
 +
Place in benchtop centrifuge at max speed for 30 seconds.</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:15.0pt;mso-bidi-font-size:12.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'><span
 +
style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span
 +
style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>7. Add 400uL endo-wash and spin at max speed on benchtop centrifuge</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:15.0pt;mso-bidi-font-size:12.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'><span
 +
style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span
 +
style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>8. Add 400uL Zippy wash buffer , centrifuge at max speed for 1
 +
minute, repeat once.</span><span style='font-size:10.0pt;font-family:Times;
 +
mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:15.0pt;mso-bidi-font-size:12.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'><span
 +
style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span
 +
style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>9. Transfer to 1.5 mL Eppendorf tube add 150uL water, let sit for
 +
1 minute before spinning at max speed for 1 minute</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;10.
 +
Check concentration with nanodrop </span><span style='font-size:10.0pt;
 +
font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br>
 +
<br style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:15.0pt;mso-bidi-font-size:12.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'><span
 +
style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>3. Transfect CB15N with plasmids to make two
 +
strains of transformed CB15N.</span><span style='font-size:10.0pt;font-family:
 +
Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>4. Grow strains on plates.</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>5. Pick colonies for overnights.</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>6. Induce fluorescence.</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>7. Check for fluorescence. </span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br>
 +
<br style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><b><span style='font-size:15.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>Gentamicin Dilution</span></b><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br
 +
style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>1. Weigh out .1g Gentamicin powder</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>2. Place in 50mL microcentrifuge tube</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>3. Add 10 mL Milli Q water</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>4. Pull plunger out of 60mL BD syringe</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>5. Screw on filter tip.</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>6. Fill syringe with solution</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>7. Empty syringe into new 50mL microcentifuge
 +
tube.</span><span style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:
 +
"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br
 +
style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><b><span style='font-size:15.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>Sterilized colony pick </span></b><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>0. Autoclave growth media in container. (allow
 +
liberal space in container for aeration of culture), after cooling, add
 +
appropriate amount of antibiotic.</span><span style='font-size:10.0pt;
 +
font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>1. Heat tungsten loop on bunsen burner until red
 +
hot.</span><span style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:
 +
"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>2. Let cool beside flame</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>3. Dip loop in agarose to check temperature</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>4. Beneath flame, carefully scrape-off one
 +
colony from plate making sure not to get any other colonies on loop.</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>5. Beneath flame, stick loop with colony in
 +
growth media making sure not to touch the sides of the container with loop.
 +
swirl around until colony washes off of loop.</span><span style='font-size:
 +
10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>6. Incubate in appropriate conditions.</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br>
 +
<br>
 +
<br>
 +
<br style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><u><span style='font-size:32.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:red'>Week 1</span></u><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>8-1-13</span><span style='font-size:10.0pt;
 +
font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<ul style='margin-top:0in' type=disc>
 +
<li class=MsoNormal style='color:black;mso-list:l14 level1 lfo1;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Made and
 +
    autoclaved PYE<o:p></o:p></span></li>
 +
<li class=MsoNormal style='color:black;mso-list:l14 level1 lfo1;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Prepared another
 +
    batch of &nbsp;electrocompetent caulobacter (culture OD600 = 0.615A)<o:p></o:p></span></li>
 +
<li class=MsoNormal style='color:black;mso-list:l14 level1 lfo1;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Induced KAN
 +
    resistant with xylose transformed caulobacter @ OD600 = 0.664A <o:p></o:p></span></li>
 +
<li class=MsoNormal style='color:black;mso-list:l14 level1 lfo1;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Non induced :
 +
    3.25ml </span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
    "Times New Roman"'>PXYFPC-2</span><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'> + CB15N, 20.5ul
 +
    antibiotic, 200ml PYE</span><span style='font-size:15.0pt;font-family:
 +
    Arial;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></li>
 +
<li class=MsoNormal style='color:black;mso-list:l14 level1 lfo1;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Induced: 3.25ml </span><span
 +
    style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman"'>PXYFPC-2</span><span
 +
    style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman"'>+
 +
    CB15N, 20.5 ul antibiotic, 200ml PYE, .4g xylose .</span><span
 +
    style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></li>
 +
<li class=MsoNormal style='color:black;mso-list:l14 level1 lfo1;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Electroporation
 +
    of CB15N with </span><span style='font-size:15.0pt;font-family:Arial;
 +
    mso-bidi-font-family:"Times New Roman"'>PVCFPC-4</span><span
 +
    style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman"'>
 +
    .</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
    "Times New Roman"'><o:p></o:p></span></li>
 +
<li class=MsoNormal style='color:black;mso-list:l14 level1 lfo1;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Plated on
 +
    PYE+agarose+gent plates 5 plates (10ul drop, 50ul drop, 100ul drop, two
 +
    negative controls (see 8-5-13))<o:p></o:p></span></li>
 +
<li class=MsoNormal style='color:black;mso-list:l14 level1 lfo1;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Plated CB15N on
 +
    KAN plate for negative control. (note : this was not prepared following
 +
    the electroporation protocol with water instead of plasmid. This was just
 +
    a drop from a colony pick shaking incubator pick of CB15N )<o:p></o:p></span></li>
 +
</ul>
 +
 +
<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>8-2-13</span></b><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<ul style='margin-top:0in' type=disc>
 +
<li class=MsoNormal style='color:black;mso-list:l9 level1 lfo2;tab-stops:list .5in;
 +
    vertical-align:baseline'><span style='font-size:16.0pt;font-family:Arial;
 +
    mso-bidi-font-family:"Times New Roman"'>Checked fluorescence of PCXFPC-2 :
 +
    UI (OD600= .76 flouereced, I OD600 = .72 no fluorescence.)<o:p></o:p></span></li>
 +
</ul>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br>
 +
<br>
 +
<br>
 +
<br>
 +
<br style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><u><span style='font-size:32.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:red'>Week 2</span></u><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>8-5-13</span></b><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<ul style='margin-top:0in' type=disc>
 +
<li class=MsoNormal style='color:black;mso-list:l16 level1 lfo3;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Checked plates <o:p></o:p></span></li>
 +
<li class=MsoNormal style='color:black;mso-list:l16 level1 lfo3;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Negative control
 +
    (CB15N+KAN electroporated with water): one colony<o:p></o:p></span></li>
 +
<li class=MsoNormal style='color:black;mso-list:l16 level1 lfo3;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Negative control
 +
    (CB15N+KAN straight from culture): many colonies<o:p></o:p></span></li>
 +
<li class=MsoNormal style='color:black;mso-list:l16 level1 lfo3;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>10uL, 50uL, and
 +
    100uL many colonies<o:p></o:p></span></li>
 +
<li class=MsoNormal style='color:black;mso-list:l16 level1 lfo3;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Result: Plates
 +
    deemed unusable<o:p></o:p></span></li>
 +
<li class=MsoNormal style='color:black;mso-list:l16 level1 lfo3;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Made new PYE +
 +
    Gentamicin plates<o:p></o:p></span></li>
 +
</ul>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br
 +
style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>8-6-13</span></b><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<ul style='margin-top:0in' type=disc>
 +
<li class=MsoNormal style='color:black;mso-list:l17 level1 lfo4;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>PXYFPC-2 +CB15N
 +
    culture to be induced made <o:p></o:p></span></li>
 +
</ul>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;mso-bidi-font-size:12.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'><span
 +
style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span
 +
style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>-5mL PXYFPC-2 + CB15N (OD600 = 0.72)</span><span style='font-size:
 +
10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<ul style='margin-top:0in' type=disc>
 +
<ul style='margin-top:0in' type=circle>
 +
  <li class=MsoNormal style='color:black;mso-list:l10 level2 lfo5;tab-stops:
 +
      list 1.0in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
      font-family:Arial;mso-bidi-font-family:"Times New Roman"'>-20uL KAN<o:p></o:p></span></li>
 +
</ul>
 +
<li class=MsoNormal style='color:black;mso-list:l10 level1 lfo5;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>50 uL PCR of
 +
    &nbsp;stpx and pflI on CB15N<o:p></o:p></span></li>
 +
<li class=MsoNormal style='color:black;mso-list:l10 level1 lfo5;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Gel
 +
    Electrophoresis on PCR reactions <o:p></o:p></span></li>
 +
</ul>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br
 +
style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>8-7-13</span></b><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<ul style='margin-top:0in' type=disc>
 +
<li class=MsoNormal style='color:black;mso-list:l22 level1 lfo6;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Transformation
 +
    of CB15N with PCXFPC-2 (electroporation) time constant = 5.6<o:p></o:p></span></li>
 +
<li class=MsoNormal style='color:black;mso-list:l22 level1 lfo6;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Transformation
 +
    of CB15N with </span><span style='font-size:15.0pt;font-family:Arial;
 +
    mso-bidi-font-family:"Times New Roman"'>PVCFPC-4</span><span
 +
    style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman"'>
 +
    (electroporation) time constant = 5.4</span><span style='font-size:15.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></li>
 +
<li class=MsoNormal style='color:black;mso-list:l22 level1 lfo6;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>PXYFPC-2 +CB15N
 +
    culture to be induced made <o:p></o:p></span></li>
 +
</ul>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;mso-bidi-font-size:12.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'><span
 +
style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span
 +
style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>-5ml PXYFPC-2 + CB15N (OD600 = 0.72)</span><span style='font-size:
 +
10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;mso-bidi-font-size:12.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'><span
 +
style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span
 +
style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>-20ul KAN</span><span style='font-size:10.0pt;font-family:Times;
 +
mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='margin-left:-12.0pt;text-indent:-.25in;mso-list:l6 level1 lfo7;
 +
tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
 +
style='font-size:10.0pt;mso-bidi-font-size:16.0pt;font-family:Symbol;
 +
mso-fareast-font-family:Symbol;mso-bidi-font-family:Symbol;color:black'><span
 +
style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
</span></span></span><![endif]><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>TD PCR for Stpx and pflI
 +
tags from caulobacter, two 15 cycle sets <o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='margin-left:-12.0pt;text-indent:-.25in;mso-list:l6 level1 lfo7;
 +
tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
 +
style='font-size:10.0pt;mso-bidi-font-size:16.0pt;font-family:Symbol;
 +
mso-fareast-font-family:Symbol;mso-bidi-font-family:Symbol;color:black'><span
 +
style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
</span></span></span><![endif]><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>Initial denature of 95 Cº<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br
 +
style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
 +
style='border-collapse:collapse'>
 +
<tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
 +
  <td valign=top style='border:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>denature
 +
  </span><span style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:
 +
  "Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>anneal</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>elongation</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>denature</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>anneal</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>elongation</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
</tr>
 +
<tr style='mso-yfti-irow:1'>
 +
  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>95
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>56
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>68
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>95
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>49
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>68
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
</tr>
 +
<tr style='mso-yfti-irow:2;mso-yfti-lastrow:yes'>
 +
  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>:10
 +
  </span><span style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:
 +
  "Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>:15
 +
  </span><span style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:
 +
  "Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>2:20</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>:10</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>:15</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>2:20</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
</tr>
 +
</table>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br>
 +
<br>
 +
<br style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><u><span style='font-size:32.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:red'>Week 3</span></u><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>8-13-13</span></b><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<ul style='margin-top:0in' type=disc>
 +
<li class=MsoNormal style='color:black;mso-list:l0 level1 lfo8;tab-stops:list .5in;
 +
    vertical-align:baseline'><span style='font-size:16.0pt;font-family:Arial;
 +
    mso-bidi-font-family:"Times New Roman"'>TD PCR for Stpx and pflI #3, 4,
 +
    and 5. Two sets of 15 cycles with initial denature at 95 C for 1:00 for #3
 +
    and 94 C for 3:00 for #4 and #5<o:p></o:p></span></li>
 +
</ul>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br
 +
style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>#3</span><span style='font-size:10.0pt;
 +
font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
 +
style='border-collapse:collapse'>
 +
<tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
 +
  <td valign=top style='border:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>denature
 +
  </span><span style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:
 +
  "Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>anneal</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>elongation</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>denature</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>anneal</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>elongation</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
</tr>
 +
<tr style='mso-yfti-irow:1'>
 +
  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>95
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>56
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>68
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>95
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>49
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>68
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
</tr>
 +
<tr style='mso-yfti-irow:2;mso-yfti-lastrow:yes'>
 +
  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>:10
 +
  </span><span style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:
 +
  "Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>:15
 +
  </span><span style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:
 +
  "Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>2:20</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>:15</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>:15</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>2:20</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
</tr>
 +
</table>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br
 +
style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>#4</span><span style='font-size:10.0pt;
 +
font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
 +
style='border-collapse:collapse'>
 +
<tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
 +
  <td valign=top style='border:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>denature
 +
  </span><span style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:
 +
  "Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>anneal</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>elongation</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>denature</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>anneal</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>elongation</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
</tr>
 +
<tr style='mso-yfti-irow:1'>
 +
  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>94
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>56
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>68
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>94
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>49
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>68
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
</tr>
 +
<tr style='mso-yfti-irow:2;mso-yfti-lastrow:yes'>
 +
  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>:15
 +
  </span><span style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:
 +
  "Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>:30
 +
  </span><span style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:
 +
  "Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>2:20</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>:15</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>:30</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>2:20</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
</tr>
 +
</table>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br
 +
style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>#5</span><span style='font-size:10.0pt;
 +
font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
 +
style='border-collapse:collapse'>
 +
<tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
 +
  <td valign=top style='border:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>denature
 +
  </span><span style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:
 +
  "Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>anneal</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>elongation</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>denature</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>anneal</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>elongation</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
</tr>
 +
<tr style='mso-yfti-irow:1'>
 +
  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>94
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>59
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>68
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>94
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>52
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>68
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
</tr>
 +
<tr style='mso-yfti-irow:2;mso-yfti-lastrow:yes'>
 +
  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>:15
 +
  </span><span style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:
 +
  "Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>:15
 +
  </span><span style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:
 +
  "Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>2:30</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>:15</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>:15</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>2:30</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
</tr>
 +
</table>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br>
 +
<br style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>8-15-13</span></b><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<ul style='margin-top:0in' type=disc>
 +
<li class=MsoNormal style='color:black;mso-list:l4 level1 lfo9;tab-stops:list .5in;
 +
    vertical-align:baseline'><span style='font-size:16.0pt;font-family:Arial;
 +
    mso-bidi-font-family:"Times New Roman"'>Made LB<o:p></o:p></span></li>
 +
<li class=MsoNormal style='color:black;mso-list:l4 level1 lfo9;tab-stops:list .5in;
 +
    vertical-align:baseline'><span style='font-size:16.0pt;font-family:Arial;
 +
    mso-bidi-font-family:"Times New Roman"'>Checked plates<o:p></o:p></span></li>
 +
</ul>
 +
 +
<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>8-19-13</span></b><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>Gentamicin dilution (10mg/mL)</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>1. Weigh out 0.1g Gentamicin powder</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>2. Place in 50mL microcentrifuge tube</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>3. Add 10 mL Milli q water</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>4. Pull plunger out of 60mL BD syringe</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>5. Screw on filter tip</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>6. Fill syringe with solution </span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>7. Empty syringe into new 50mL microcentrifuge
 +
tube.</span><span style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:
 +
"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>8.aliquot 100uL, 10uL, 500uL, and 10uL into
 +
1.5ml Eppendorf tubes</span><span style='font-size:10.0pt;font-family:Times;
 +
mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br
 +
style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<ul style='margin-top:0in' type=disc>
 +
<li class=MsoNormal style='color:black;mso-list:l3 level1 lfo10;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Sterilized
 +
    inoculation of LB with <i>E. coli</i> + PXYFPC-2 +KAN (30ug/mL final
 +
    concentration)<o:p></o:p></span></li>
 +
<li class=MsoNormal style='color:black;mso-list:l3 level1 lfo10;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Sterilized
 +
    inoculation of LB with <i>E. coli </i>+ PVCFPC-4 +GENT (15ug/mL final
 +
    concentration)<o:p></o:p></span></li>
 +
</ul>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br
 +
style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>8-20-13</span></b><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>Created Glycerol stock of <i>E. coli</i>
 +
cultures containing PVCFPC-4</span><span style='font-size:10.0pt;font-family:
 +
Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>Plasmid Mini-prep of<i> E. col</i>i cultures</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='margin-left:-12.0pt;text-indent:-.25in;mso-list:l19 level1 lfo11;
 +
tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
 +
style='font-size:10.0pt;mso-bidi-font-size:16.0pt;font-family:Symbol;
 +
mso-fareast-font-family:Symbol;mso-bidi-font-family:Symbol;color:black'><span
 +
style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
</span></span></span><![endif]><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>Culture #1 OD550 .916<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='margin-left:-12.0pt;text-indent:-.25in;mso-list:l19 level1 lfo11;
 +
tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
 +
style='font-size:10.0pt;mso-bidi-font-size:16.0pt;font-family:Symbol;
 +
mso-fareast-font-family:Symbol;mso-bidi-font-family:Symbol;color:black'><span
 +
style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
</span></span></span><![endif]><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>Culture #2 OD5050 2.149<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br
 +
style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
 +
style='border-collapse:collapse'>
 +
<tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
 +
  <td valign=top style='border:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  10.0pt;font-family:Times'><br style='mso-special-character:line-break'>
 +
  <![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
  <![endif]><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>Nucleic
 +
  Acid Concentration</span><span style='font-size:10.0pt;font-family:Times;
 +
  mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>A260</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>A280</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>260/280</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>260/230</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
</tr>
 +
<tr style='mso-yfti-irow:1'>
 +
  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>Culture
 +
  #1</span><span style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:
 +
  "Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>21.0
 +
  ng/uL</span><span style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:
 +
  "Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>.419</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>.221</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>1.89</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>1.68</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
</tr>
 +
<tr style='mso-yfti-irow:2;mso-yfti-lastrow:yes'>
 +
  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>Culture
 +
  #2</span><span style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:
 +
  "Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>299.2
 +
  ng/uL</span><span style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:
 +
  "Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>5.983</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>3.184</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>1.88</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>2.13</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
</tr>
 +
</table>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br
 +
style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><u><span style='font-size:32.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:red'>Week 5</span></u><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>9-?-13</span></b><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<ul style='margin-top:0in' type=disc>
 +
<li class=MsoNormal style='color:black;mso-list:l20 level1 lfo12;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Use Geneious to
 +
    design primers (forward and reverse) to get Halorhodopsin (HsHR) out of <i>H.Salinarum.</i><o:p></o:p></span></li>
 +
<li class=MsoNormal style='color:black;mso-list:l20 level1 lfo12;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Order primers.<o:p></o:p></span></li>
 +
</ul>
 +
 +
<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>9-3-13</span></b><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<ul style='margin-top:0in' type=disc>
 +
<li class=MsoNormal style='color:black;mso-list:l15 level1 lfo13;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Take chip of
 +
    frozen glycerol stock H.Salinarum and suspend in Milli Q Water<o:p></o:p></span></li>
 +
</ul>
 +
 +
<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>9-4-13</span></b><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>25 uL PCR using HsHR primers ,HS (template), and
 +
GC master mix.</span><span style='font-size:10.0pt;font-family:Times;
 +
mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<ul style='margin-top:0in' type=disc>
 +
<li class=MsoNormal style='color:black;mso-list:l13 level1 lfo14;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Negative control
 +
    without primers labeled (-1)<o:p></o:p></span></li>
 +
<li class=MsoNormal style='color:black;mso-list:l13 level1 lfo14;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Negative control
 +
    without HS (template) labeled (-2)<o:p></o:p></span></li>
 +
<li class=MsoNormal style='color:black;mso-list:l13 level1 lfo14;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>98C</span><span
 +
    style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman"'>º</span><span
 +
    style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman"'>
 +
    -2mins/98C</span><span style='font-size:15.0pt;font-family:Arial;
 +
    mso-bidi-font-family:"Times New Roman"'>º</span><span style='font-size:
 +
    16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman"'> 10s 57.2C</span><span
 +
    style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman"'>º</span><span
 +
    style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman"'>
 +
    72C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
    "Times New Roman"'>º</span><span style='font-size:16.0pt;font-family:Arial;
 +
    mso-bidi-font-family:"Times New Roman"'> 2 min/72C</span><span
 +
    style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman"'>º</span><span
 +
    style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman"'>
 +
    3 min hold &nbsp;4C</span><span style='font-size:15.0pt;font-family:Arial;
 +
    mso-bidi-font-family:"Times New Roman"'>º<o:p></o:p></span></li>
 +
</ul>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br>
 +
<br>
 +
<br style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>9-5-13</span></b><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<ul style='margin-top:0in' type=disc>
 +
<li class=MsoNormal style='color:black;mso-list:l21 level1 lfo15;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Sterilized
 +
    inoculation of SOC &nbsp;with iGEM part BBa_K9000 “9000” + <i>E. coli</i><o:p></o:p></span></li>
 +
<li class=MsoNormal style='color:black;mso-list:l21 level1 lfo15;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Sterilized
 +
    inoculation of SOC with iGEM part BBa_K9001 “9001” + <i>E. coli</i><o:p></o:p></span></li>
 +
<li class=MsoNormal style='color:black;mso-list:l21 level1 lfo15;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Sterilized
 +
    inoculation of SOC with iGEM part BBa_K9010 “9010” + <i>E. coli</i><o:p></o:p></span></li>
 +
<li class=MsoNormal style='color:black;mso-list:l21 level1 lfo15;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Made
 +
    chloramphenicol (1ug/1mL) plates<o:p></o:p></span></li>
 +
</ul>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br
 +
style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>PCR of Halorhodopsin from Halobacterium
 +
Salinarum 30 cycles</span><span style='font-size:10.0pt;font-family:Times;
 +
mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>25 uL reaction with 2x Phusion GC Master Mix</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br
 +
style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
 +
style='border-collapse:collapse'>
 +
<tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
 +
  <td valign=top style='border:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>initial
 +
  denature</span><span style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:
 +
  "Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>denature</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>anneal</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>extension</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>final
 +
  extension</span><span style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:
 +
  "Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
</tr>
 +
<tr style='mso-yfti-irow:1'>
 +
  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>98
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>98
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>57
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>72
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>72
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
</tr>
 +
<tr style='mso-yfti-irow:2;mso-yfti-lastrow:yes'>
 +
  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>2:00</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>00:10
 +
  </span><span style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:
 +
  "Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>00:20</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>1:00</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>5:00</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
</tr>
 +
</table>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br>
 +
<span style='mso-no-proof:yes'><!--[if gte vml 1]><v:shapetype id="_x0000_t75"
 +
coordsize="21600,21600" o:spt="75" o:preferrelative="t" path="m@4@5l@4@11@9@11@9@5xe"
 +
filled="f" stroked="f">
 +
<v:stroke joinstyle="miter"/>
 +
<v:formulas>
 +
  <v:f eqn="if lineDrawn pixelLineWidth 0"/>
 +
  <v:f eqn="sum @0 1 0"/>
 +
  <v:f eqn="sum 0 0 @1"/>
 +
  <v:f eqn="prod @2 1 2"/>
 +
  <v:f eqn="prod @3 21600 pixelWidth"/>
 +
  <v:f eqn="prod @3 21600 pixelHeight"/>
 +
  <v:f eqn="sum @0 0 1"/>
 +
  <v:f eqn="prod @6 1 2"/>
 +
  <v:f eqn="prod @7 21600 pixelWidth"/>
 +
  <v:f eqn="sum @8 21600 0"/>
 +
  <v:f eqn="prod @7 21600 pixelHeight"/>
 +
  <v:f eqn="sum @10 21600 0"/>
 +
</v:formulas>
 +
<v:path o:extrusionok="f" gradientshapeok="t" o:connecttype="rect"/>
 +
<o:lock v:ext="edit" aspectratio="t"/>
 +
</v:shapetype><v:shape id="Picture_x0020_1" o:spid="_x0000_i1034" type="#_x0000_t75"
 +
alt="https://lh5.googleusercontent.com/zoqmY8_jwDO6H4kdmXWKQDbayg_t_7tTOV2xuIHnLhfuI6g5P6IAC4aixAv79aV4FzsEX_GDs2rTSGPB9YVESOBxO_10EBqNa3XHbhmCxGzjC2mL5S14MKY_-g2gULexjho"
 +
style='width:732pt;height:874pt;visibility:visible;mso-wrap-style:square'>
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<v:imagedata src="Pumpsnotebook_files/image001.jpg" o:title="//lh5.googleusercontent.com/zoqmY8_jwDO6H4kdmXWKQDbayg_t_7tTOV2xuIHnLhfuI6g5P6IAC4aixAv79aV4FzsEX_GDs2rTSGPB9YVESOBxO_10EBqNa3XHbhmCxGzjC2mL5S14MKY_-g2gULexjho"/>
 +
<v:textbox style='mso-rotate-with-shape:t'/>
 +
</v:shape><![endif]--><![if !vml]><img width=734 height=876
 +
src="Pumpsnotebook_files/image002.jpg"
 +
alt="https://lh5.googleusercontent.com/zoqmY8_jwDO6H4kdmXWKQDbayg_t_7tTOV2xuIHnLhfuI6g5P6IAC4aixAv79aV4FzsEX_GDs2rTSGPB9YVESOBxO_10EBqNa3XHbhmCxGzjC2mL5S14MKY_-g2gULexjho"
 +
v:shapes="Picture_x0020_1"><![endif]></span><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>9-6-13</span><span style='font-size:10.0pt;
 +
font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>Plasmid miniprep on 9000, 9010, and 9010 cultures.</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;mso-bidi-font-size:12.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'><span
 +
style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span
 +
style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>Results: </span><span style='font-size:10.0pt;font-family:Times;
 +
mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;mso-bidi-font-size:12.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'><span
 +
style='mso-tab-count:2'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span
 +
style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>-9000 = 99.2 ng/ml</span><span style='font-size:10.0pt;font-family:
 +
Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;mso-bidi-font-size:12.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'><span
 +
style='mso-tab-count:2'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span
 +
style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>-9001 =99.7 ng/ml</span><span style='font-size:10.0pt;font-family:
 +
Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;mso-bidi-font-size:12.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'><span
 +
style='mso-tab-count:2'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span
 +
style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>-9010 = 38.1 ng/ml</span><span style='font-size:10.0pt;font-family:
 +
Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;mso-bidi-font-size:12.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'><span
 +
style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span
 +
style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>Gel legend : 1Kb ladder , 9010, 9001, 9000</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<ul style='margin-top:0in' type=disc>
 +
<li class=MsoNormal style='color:black;mso-list:l2 level1 lfo16;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Design and order
 +
    sequencing primers for iGEM parts. same forward and reverse for all three.<o:p></o:p></span></li>
 +
</ul>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br
 +
style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><u><span style='font-size:32.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:red'>Week 6</span></u><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>9-9-13</span></b><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<ul style='margin-top:0in' type=disc>
 +
<li class=MsoNormal style='color:black;mso-list:l12 level1 lfo17;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Send plasmids
 +
    with sequencing primers out for sequencing.<o:p></o:p></span></li>
 +
<li class=MsoNormal style='color:black;mso-list:l12 level1 lfo17;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Design and order
 +
    primers for plasmid backbone.<o:p></o:p></span></li>
 +
<li class=MsoNormal style='color:black;mso-list:l12 level1 lfo17;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>20uL PCR on HS
 +
    for HsHR<o:p></o:p></span></li>
 +
<li class=MsoNormal style='color:black;mso-list:l12 level1 lfo17;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Run diagnostic
 +
    gel <o:p></o:p></span></li>
 +
</ul>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;mso-bidi-font-size:12.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'><span
 +
style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span
 +
style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>- Gel legend : 1Kb ladder , H? , +, -1, -2, </span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_2" o:spid="_x0000_i1033"
 +
type="#_x0000_t75" alt="https://lh5.googleusercontent.com/0Q85u88g6Sx3Xg9d2cGCLZjT4g7NqocTnSygFFPr4x_linJjMLnbRbHAI205lmA6Xj8KPv1-SCKYuEaqDhQxhe3hXULLqb4k1cWU7cLtNqLUiWHi1Q7nLKW58hr10V6UVsM"
 +
style='width:1600pt;height:1200pt;visibility:visible;mso-wrap-style:square'>
 +
<v:imagedata src="Pumpsnotebook_files/image003.jpg" o:title="//lh5.googleusercontent.com/0Q85u88g6Sx3Xg9d2cGCLZjT4g7NqocTnSygFFPr4x_linJjMLnbRbHAI205lmA6Xj8KPv1-SCKYuEaqDhQxhe3hXULLqb4k1cWU7cLtNqLUiWHi1Q7nLKW58hr10V6UVsM"/>
 +
<v:textbox style='mso-rotate-with-shape:t'/>
 +
</v:shape><![endif]--><![if !vml]><img width=1602 height=1202
 +
src="Pumpsnotebook_files/image004.jpg"
 +
alt="https://lh5.googleusercontent.com/0Q85u88g6Sx3Xg9d2cGCLZjT4g7NqocTnSygFFPr4x_linJjMLnbRbHAI205lmA6Xj8KPv1-SCKYuEaqDhQxhe3hXULLqb4k1cWU7cLtNqLUiWHi1Q7nLKW58hr10V6UVsM"
 +
v:shapes="Picture_x0020_2"><![endif]></span><span style='font-size:10.0pt;
 +
font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>9-10-13</span></b><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<ul style='margin-top:0in' type=disc>
 +
<li class=MsoNormal style='color:black;mso-list:l1 level1 lfo18;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>50 ul HsHR PCR
 +
    labeled “50”<o:p></o:p></span></li>
 +
<li class=MsoNormal style='color:black;mso-list:l1 level1 lfo18;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>98C 2min/ 98C</span><span
 +
    style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman"'>º</span><span
 +
    style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman"'>
 +
    :10s/57.2C</span><span style='font-size:15.0pt;font-family:Arial;
 +
    mso-bidi-font-family:"Times New Roman"'>º</span><span style='font-size:
 +
    16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman"'> 72C</span><span
 +
    style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman"'>º</span><span
 +
    style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman"'>
 +
    1min/72min/Hold 4C</span><span style='font-size:15.0pt;font-family:Arial;
 +
    mso-bidi-font-family:"Times New Roman"'>º</span><span style='font-size:
 +
    16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></li>
 +
</ul>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br>
 +
<br>
 +
<span style='mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_3"
 +
o:spid="_x0000_i1032" type="#_x0000_t75" alt="https://lh5.googleusercontent.com/z0GTplt-8fYo_yphibg41YCiunGWerxVFXSEPXuFcTzv9dRwAsJka3syaRQ6oM_koPX44Tejz8JeQvTLEbnaX2aCEQh0mOmYbmQ4D-drnfb-uudDOYdeSeUJQdltHoqZjNk"
 +
style='width:695pt;height:839pt;visibility:visible;mso-wrap-style:square'>
 +
<v:imagedata src="Pumpsnotebook_files/image005.jpg" o:title="//lh5.googleusercontent.com/z0GTplt-8fYo_yphibg41YCiunGWerxVFXSEPXuFcTzv9dRwAsJka3syaRQ6oM_koPX44Tejz8JeQvTLEbnaX2aCEQh0mOmYbmQ4D-drnfb-uudDOYdeSeUJQdltHoqZjNk"/>
 +
<v:textbox style='mso-rotate-with-shape:t'/>
 +
</v:shape><![endif]--><![if !vml]><img width=697 height=841
 +
src="Pumpsnotebook_files/image006.jpg"
 +
alt="https://lh5.googleusercontent.com/z0GTplt-8fYo_yphibg41YCiunGWerxVFXSEPXuFcTzv9dRwAsJka3syaRQ6oM_koPX44Tejz8JeQvTLEbnaX2aCEQh0mOmYbmQ4D-drnfb-uudDOYdeSeUJQdltHoqZjNk"
 +
v:shapes="Picture_x0020_3"><![endif]></span><br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>9-11-13</span></b><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>Purification gel</span><span style='font-size:
 +
10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;mso-bidi-font-size:12.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'><span
 +
style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span
 +
style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>-Gel Legend: 1Kb ladder, 50</span><span style='font-size:10.0pt;
 +
font-family:Times;mso-bidi-font-family:"Times New Roman";mso-no-proof:yes'><!--[if gte vml 1]><v:shape
 +
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 +
style='width:575pt;height:639pt;visibility:visible;mso-wrap-style:square'>
 +
<v:imagedata src="Pumpsnotebook_files/image007.jpg" o:title="//lh6.googleusercontent.com/kZa_3xX536Vv51c7eTVUpKXaR6GQmOhwebCL2oxD8a3m68bfWNXnZGquK-EY-x9CmYC4UUaajzn6qJPtGWfkKRh5WD-KHChocRyNonwjwdh7y_9RrlKDY68eHNND1Gf1zRk"/>
 +
<v:textbox style='mso-rotate-with-shape:t'/>
 +
</v:shape><![endif]--><![if !vml]><img width=577 height=641
 +
src="Pumpsnotebook_files/image008.jpg"
 +
alt="https://lh6.googleusercontent.com/kZa_3xX536Vv51c7eTVUpKXaR6GQmOhwebCL2oxD8a3m68bfWNXnZGquK-EY-x9CmYC4UUaajzn6qJPtGWfkKRh5WD-KHChocRyNonwjwdh7y_9RrlKDY68eHNND1Gf1zRk"
 +
v:shapes="Picture_x0020_4"><![endif]></span><span style='font-size:10.0pt;
 +
font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<ul style='margin-top:0in' type=disc>
 +
<li class=MsoNormal style='color:black;mso-list:l23 level1 lfo19;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>50ul HsHR PCR
 +
    (two reactions “50(1)” and “50(2)”) one negative control with no template
 +
    “50 -”<o:p></o:p></span></li>
 +
</ul>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>Gel Legend : 1Kb ladder , 50(1), 50(2), 50-</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_5" o:spid="_x0000_i1030"
 +
type="#_x0000_t75" alt="https://lh3.googleusercontent.com/zKPXvbXlR8t8qYT3XrTzaQkip94KqBpkA8Y4Cdflat05xNktHf1q-RY5GRdGIiB20p7SwrDf0V0cLxhjREvwIlVZ_h_19M-JuvjjtWwLoOR-JHBInZhkqeEcX321Qwd3SYY"
 +
style='width:443pt;height:563pt;visibility:visible;mso-wrap-style:square'>
 +
<v:imagedata src="Pumpsnotebook_files/image009.jpg" o:title="//lh3.googleusercontent.com/zKPXvbXlR8t8qYT3XrTzaQkip94KqBpkA8Y4Cdflat05xNktHf1q-RY5GRdGIiB20p7SwrDf0V0cLxhjREvwIlVZ_h_19M-JuvjjtWwLoOR-JHBInZhkqeEcX321Qwd3SYY"/>
 +
<v:textbox style='mso-rotate-with-shape:t'/>
 +
</v:shape><![endif]--><![if !vml]><img width=445 height=565
 +
src="Pumpsnotebook_files/image010.jpg"
 +
alt="https://lh3.googleusercontent.com/zKPXvbXlR8t8qYT3XrTzaQkip94KqBpkA8Y4Cdflat05xNktHf1q-RY5GRdGIiB20p7SwrDf0V0cLxhjREvwIlVZ_h_19M-JuvjjtWwLoOR-JHBInZhkqeEcX321Qwd3SYY"
 +
v:shapes="Picture_x0020_5"><![endif]></span><span style='font-size:10.0pt;
 +
font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br
 +
style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>9-12-13</span></b><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>50uL PCR to obtain plasmid backbone using 9010
 +
(30.4ng/ul) as template. </span><span style='font-size:10.0pt;font-family:Times;
 +
mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br
 +
style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
 +
style='border-collapse:collapse'>
 +
<tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
 +
  <td valign=top style='border:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>initial
 +
  denature</span><span style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:
 +
  "Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>denature</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>anneal</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>extension</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border:solid black 1.0pt;border-left:none;mso-border-left-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>final
 +
  extension</span><span style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:
 +
  "Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
</tr>
 +
<tr style='mso-yfti-irow:1'>
 +
  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>98
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>98
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>52.2
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>72
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>72
 +
  C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
  "Times New Roman";color:black'>º</span><span style='font-size:10.0pt;
 +
  font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
</tr>
 +
<tr style='mso-yfti-irow:2;mso-yfti-lastrow:yes'>
 +
  <td valign=top style='border:solid black 1.0pt;border-top:none;mso-border-top-alt:
 +
  solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>:30</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>:10</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>:30</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>1:00</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
  <td valign=top style='border-top:none;border-left:none;border-bottom:solid black 1.0pt;
 +
  border-right:solid black 1.0pt;mso-border-top-alt:solid black 1.0pt;
 +
  mso-border-left-alt:solid black 1.0pt;padding:7.0pt 7.0pt 7.0pt 7.0pt'>
 +
  <p class=MsoNormal style='mso-line-height-alt:0pt'><span style='font-size:
 +
  16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>5:00</span><span
 +
  style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
  </td>
 +
</tr>
 +
</table>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>Gel Legend : 1Kb ladder , 50(1), 50(2), 50-</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_6" o:spid="_x0000_i1029"
 +
type="#_x0000_t75" alt="https://lh4.googleusercontent.com/LraY7dEsn1ebWvlutcCKsiU9hRs7QVvUkVoi7SedZHzyHJm1j0gWpDeYX3E7mYUY2UhKTL3t9dvCI-IOgIcxvTz9FWQQBr2iIHIs7jykiD0QyCtjvLXO31xbJxwWUsZ58fo"
 +
style='width:713pt;height:838pt;visibility:visible;mso-wrap-style:square'>
 +
<v:imagedata src="Pumpsnotebook_files/image011.jpg" o:title="//lh4.googleusercontent.com/LraY7dEsn1ebWvlutcCKsiU9hRs7QVvUkVoi7SedZHzyHJm1j0gWpDeYX3E7mYUY2UhKTL3t9dvCI-IOgIcxvTz9FWQQBr2iIHIs7jykiD0QyCtjvLXO31xbJxwWUsZ58fo"/>
 +
<v:textbox style='mso-rotate-with-shape:t'/>
 +
</v:shape><![endif]--><![if !vml]><img width=715 height=840
 +
src="Pumpsnotebook_files/image012.jpg"
 +
alt="https://lh4.googleusercontent.com/LraY7dEsn1ebWvlutcCKsiU9hRs7QVvUkVoi7SedZHzyHJm1j0gWpDeYX3E7mYUY2UhKTL3t9dvCI-IOgIcxvTz9FWQQBr2iIHIs7jykiD0QyCtjvLXO31xbJxwWUsZ58fo"
 +
v:shapes="Picture_x0020_6"><![endif]></span><span style='font-size:10.0pt;
 +
font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br
 +
style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>Two 20uL PCR reactions using part 9010 with different
 +
annealing temperatures. One at 55 C</span><span style='font-size:15.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>º</span><span
 +
style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'> and one at 57 C</span><span style='font-size:15.0pt;font-family:
 +
Arial;mso-bidi-font-family:"Times New Roman";color:black'>º</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>Gel Legend : 1Kb ladder , 9010(55 C</span><span
 +
style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>º</span><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>) , 9010 neg (55 C</span><span
 +
style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>º</span><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>), 9010 (57 C</span><span
 +
style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>º</span><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>), 9010 neg (57C</span><span
 +
style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>º</span><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>) </span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br>
 +
<span style='mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_7"
 +
o:spid="_x0000_i1028" type="#_x0000_t75" alt="https://lh3.googleusercontent.com/5K0qLR04DEe-T0RhHNMtSAFiqwcXA7GKVEWPKcMez4AvAJkSmL1mP_0C6c84miYtFQFl4iy4y2qNe0EXgmQpTfz4eklCrLgmCc3eSES065g2q0E24XjDi_FghZp-eusnbiM"
 +
style='width:728pt;height:832pt;visibility:visible;mso-wrap-style:square'>
 +
<v:imagedata src="Pumpsnotebook_files/image013.jpg" o:title="//lh3.googleusercontent.com/5K0qLR04DEe-T0RhHNMtSAFiqwcXA7GKVEWPKcMez4AvAJkSmL1mP_0C6c84miYtFQFl4iy4y2qNe0EXgmQpTfz4eklCrLgmCc3eSES065g2q0E24XjDi_FghZp-eusnbiM"/>
 +
<v:textbox style='mso-rotate-with-shape:t'/>
 +
</v:shape><![endif]--><![if !vml]><img width=730 height=834
 +
src="Pumpsnotebook_files/image014.jpg"
 +
alt="https://lh3.googleusercontent.com/5K0qLR04DEe-T0RhHNMtSAFiqwcXA7GKVEWPKcMez4AvAJkSmL1mP_0C6c84miYtFQFl4iy4y2qNe0EXgmQpTfz4eklCrLgmCc3eSES065g2q0E24XjDi_FghZp-eusnbiM"
 +
v:shapes="Picture_x0020_7"><![endif]></span><br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>9-14-13</span></b><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>20uL PCR to obtain plasmid backbone using
 +
linearized backbone as template.</span><span style='font-size:10.0pt;
 +
font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br
 +
style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>Gel Legend : 1Kb ladder , 9010 57 C , 9010 57
 +
C(-), &nbsp;linear 57, linear 57 neg </span><span style='font-size:10.0pt;
 +
font-family:Times;mso-bidi-font-family:"Times New Roman";mso-no-proof:yes'><!--[if gte vml 1]><v:shape
 +
id="Picture_x0020_8" o:spid="_x0000_i1027" type="#_x0000_t75" alt="https://lh3.googleusercontent.com/Tsl3MNPOOovCvXGUArAeLgUvmGzZIqgHq0tkelEfgozwCS50xtEk-w9WaLuvW_mw-LA7MCNzxXrLJT8ErcyEsBwv2Ez2unP6-C5QxlcKnbUYH8Lo3qVvp3yVRgHsrEt9LLA"
 +
style='width:721pt;height:808pt;visibility:visible;mso-wrap-style:square'>
 +
<v:imagedata src="Pumpsnotebook_files/image015.jpg" o:title="//lh3.googleusercontent.com/Tsl3MNPOOovCvXGUArAeLgUvmGzZIqgHq0tkelEfgozwCS50xtEk-w9WaLuvW_mw-LA7MCNzxXrLJT8ErcyEsBwv2Ez2unP6-C5QxlcKnbUYH8Lo3qVvp3yVRgHsrEt9LLA"/>
 +
<v:textbox style='mso-rotate-with-shape:t'/>
 +
</v:shape><![endif]--><![if !vml]><img width=723 height=810
 +
src="Pumpsnotebook_files/image016.jpg"
 +
alt="https://lh3.googleusercontent.com/Tsl3MNPOOovCvXGUArAeLgUvmGzZIqgHq0tkelEfgozwCS50xtEk-w9WaLuvW_mw-LA7MCNzxXrLJT8ErcyEsBwv2Ez2unP6-C5QxlcKnbUYH8Lo3qVvp3yVRgHsrEt9LLA"
 +
v:shapes="Picture_x0020_8"><![endif]></span><span style='font-size:10.0pt;
 +
font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><u><span style='font-size:32.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:red'>Week 7</span></u><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>9-15-13</span></b><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<ul style='margin-top:0in' type=disc>
 +
<li class=MsoNormal style='color:black;mso-list:l11 level1 lfo20;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>New PCR of 9010
 +
    at 55 C</span><span style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:
 +
    "Times New Roman"'>º</span><span style='font-size:16.0pt;font-family:Arial;
 +
    mso-bidi-font-family:"Times New Roman"'> and 57 C</span><span
 +
    style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman"'>º</span><span
 +
    style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman"'>
 +
    with new GC phusion mastermix due to contamination</span><span
 +
    style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></li>
 +
<li class=MsoNormal style='color:black;mso-list:l11 level1 lfo20;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>a PCR of Linear
 +
    PSB1C3 at 57 C</span><span style='font-size:15.0pt;font-family:Arial;
 +
    mso-bidi-font-family:"Times New Roman"'>º</span><span style='font-size:
 +
    16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></li>
 +
</ul>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br
 +
style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>gel legend : 9010(55+), 9010(55 C-) , 9010(57
 +
C+), 9010(57 C-), Linear (1), Linear (2), Linear(-), DNA ladder</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><!--[if gte vml 1]><v:shape
 +
id="_x0000_i1025" type="#_x0000_t75" alt="" style='width:24pt;height:24pt'/><![endif]--><![if !vml]><img
 +
width=26 height=26 src="Pumpsnotebook_files/image017.png" v:shapes="_x0000_i1025"><![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br
 +
style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>9-16-13</span></b><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>Gel purification on PCR with linearized backbone
 +
</span><span style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:
 +
"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br
 +
style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='margin-left:-12.0pt;text-indent:-.25in;mso-list:l8 level1 lfo21;
 +
tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
 +
style='font-size:10.0pt;mso-bidi-font-size:16.0pt;font-family:Symbol;
 +
mso-fareast-font-family:Symbol;mso-bidi-font-family:Symbol;color:black'><span
 +
style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
</span></span></span><![endif]><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>Run two samples from the
 +
PCR reaction, one with 5uL pcr and loading buffer with Sybr gold, and the other
 +
with the rest of the PCR (45uL) and loading buffer without Sybr gold.<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='margin-left:-12.0pt;text-indent:-.25in;mso-list:l8 level1 lfo21;
 +
tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
 +
style='font-size:10.0pt;mso-bidi-font-size:16.0pt;font-family:Symbol;
 +
mso-fareast-font-family:Symbol;mso-bidi-font-family:Symbol;color:black'><span
 +
style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
</span></span></span><![endif]><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>Load the two samples in
 +
side by side lanes<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='margin-left:-12.0pt;text-indent:-.25in;mso-list:l8 level1 lfo21;
 +
tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
 +
style='font-size:10.0pt;mso-bidi-font-size:16.0pt;font-family:Symbol;
 +
mso-fareast-font-family:Symbol;mso-bidi-font-family:Symbol;color:black'><span
 +
style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
</span></span></span><![endif]><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>Use the Sybr gold in uv
 +
light to find the band to cut out of the gel in the next lane over.<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='margin-left:-12.0pt;text-indent:-.25in;mso-list:l8 level1 lfo21;
 +
tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
 +
style='font-size:10.0pt;mso-bidi-font-size:16.0pt;font-family:Symbol;
 +
mso-fareast-font-family:Symbol;mso-bidi-font-family:Symbol;color:black'><span
 +
style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
</span></span></span><![endif]><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>Centrifuge gel cut out with
 +
gel filter to separate. Collect filtrate.<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='margin-left:-12.0pt;text-indent:-.25in;mso-list:l8 level1 lfo21;
 +
tab-stops:list .5in;vertical-align:baseline'><![if !supportLists]><span
 +
style='font-size:10.0pt;mso-bidi-font-size:16.0pt;font-family:Symbol;
 +
mso-fareast-font-family:Symbol;mso-bidi-font-family:Symbol;color:black'><span
 +
style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
</span></span></span><![endif]><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>Use Zymo research DNA Clean
 +
and Concentrate to recover DNA from filtrate<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br
 +
style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>9-17-13</span></b><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>Concentrate DNA</span><span style='font-size:
 +
10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>1. Combine all gel purification tubes with
 +
purified DNA (add alcohol?)</span><span style='font-size:10.0pt;font-family:
 +
Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>2. Place separate cap with holes in it on top of
 +
tube.</span><span style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:
 +
"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>3. Place tube in brick under vacuum chamber.</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>4. When all liquid is gone, add 5uL of Milli Q
 +
water.</span><span style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:
 +
"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br
 +
style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>9-18-13</span></b><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>Gibson reaction with KR2 G-blocks</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;mso-bidi-font-size:12.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'><span
 +
style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span
 +
style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>-5uL concentrated plasmid backbone</span><span style='font-size:
 +
10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;mso-bidi-font-size:12.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'><span
 +
style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span
 +
style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>-1uL of each G-block</span><span style='font-size:10.0pt;
 +
font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;mso-bidi-font-size:12.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'><span
 +
style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span
 +
style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>-3uL H2O</span><span style='font-size:10.0pt;font-family:Times;
 +
mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;mso-bidi-font-size:12.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'><span
 +
style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span
 +
style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>-10uL 2x Gibson Master Mix</span><span style='font-size:10.0pt;
 +
font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;mso-bidi-font-size:12.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'><span
 +
style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><span
 +
style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>-PCR Machine 50C</span><span style='font-size:15.0pt;font-family:
 +
Arial;mso-bidi-font-family:"Times New Roman";color:black'>º</span><span
 +
style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'> for 1 hr.</span><span style='font-size:10.0pt;font-family:Times;
 +
mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>Chemi-competent transformation of <i>E.coli</i>
 +
with Gibson reaction products.</span><span style='font-size:10.0pt;font-family:
 +
Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<ul style='margin-top:0in' type=disc>
 +
<li class=MsoNormal style='color:black;mso-list:l18 level1 lfo22;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Plate transformed<i>
 +
    E.coli</i> on LB+Chloramphenicol plates. (400uL, 200uL, 100uL, and one
 +
    with untransformed <i>E. coli</i>)<o:p></o:p></span></li>
 +
</ul>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br>
 +
<br style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>9-19-13</span></b><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>Suspend colonies in Milli Q water</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<ul style='margin-top:0in' type=disc>
 +
<li class=MsoNormal style='color:black;mso-list:l7 level1 lfo23;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>Milli Q water
 +
    with colonies colony PCR on colonies from 400uL and 200uL plates<o:p></o:p></span></li>
 +
<li class=MsoNormal style='color:black;mso-list:l7 level1 lfo23;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>15 cycles<o:p></o:p></span></li>
 +
</ul>
 +
 +
<p class=MsoNormal style='text-indent:.5in'><span style='font-size:16.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>- 94 C</span><span
 +
style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>º</span><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'> initial denature 5 minutes</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='text-indent:.5in'><span style='font-size:16.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>- 94 C</span><span
 +
style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>º</span><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'> denature 15 s</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='text-indent:.5in'><span style='font-size:16.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>- 55 C</span><span
 +
style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>º</span><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'> first anneal 30s</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='text-indent:.5in'><span style='font-size:16.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>- 68 C</span><span
 +
style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>º</span><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'> extension 1:00</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br
 +
style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<ul style='margin-top:0in' type=disc>
 +
<li class=MsoNormal style='color:black;mso-list:l5 level1 lfo24;tab-stops:
 +
    list .5in;vertical-align:baseline'><span style='font-size:16.0pt;
 +
    font-family:Arial;mso-bidi-font-family:"Times New Roman"'>15 cycles anneal
 +
    drop down 7 C</span><span style='font-size:15.0pt;font-family:Arial;
 +
    mso-bidi-font-family:"Times New Roman"'>º<o:p></o:p></span></li>
 +
</ul>
 +
 +
<p class=MsoNormal style='text-indent:.5in'><span style='font-size:16.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>- 94 C</span><span
 +
style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>º</span><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'> denature :15</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='text-indent:.5in'><span style='font-size:16.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>- 48 C</span><span
 +
style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>º</span><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'> second anneal &nbsp;:30</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='text-indent:.5in'><span style='font-size:16.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>- 68 C</span><span
 +
style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>º</span><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'> extension 1:00</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='text-indent:.5in'><span style='font-size:16.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>- 68 C</span><span
 +
style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>º</span><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'> final extension 5:00</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='text-indent:.5in'><span style='font-size:16.0pt;
 +
font-family:Arial;mso-bidi-font-family:"Times New Roman";color:black'>- 4 C</span><span
 +
style='font-size:15.0pt;font-family:Arial;mso-bidi-font-family:"Times New Roman";
 +
color:black'>º</span><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'> hold infinite</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br
 +
style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>Inoculation of <i>E. coli</i> colonies in LB +
 +
chloramphenicol </span><span style='font-size:10.0pt;font-family:Times;
 +
mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br>
 +
<br style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:Arial;
 +
mso-bidi-font-family:"Times New Roman";color:black'>9-20-13</span></b><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:10.0pt;font-family:Times'><br
 +
style='mso-special-character:line-break'>
 +
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
 +
<![endif]><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>Run gel on PCR products from 9-19-13</span><span
 +
style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>Gel Legend: 1Kb ladder, “400”, “200”, negative
 +
control (no template)</span><span style='font-size:10.0pt;font-family:Times;
 +
mso-bidi-font-family:"Times New Roman";mso-no-proof:yes'><!--[if gte vml 1]><v:shape
 +
id="Picture_x0020_10" o:spid="_x0000_i1026" type="#_x0000_t75" alt="https://lh6.googleusercontent.com/TQo0ALhzurvftLHK7JHJU21cHEF0B1SYa5t5HMs02gDfTiVbi9-PxpcCEqaaB5gAJh-_bK15m9Gm1Y5wcnJfmdt-afUAcNYNhVBR9IWiQcGw0w4eB7t_YHcwi5lDPT2bhgE"
 +
style='width:683pt;height:817pt;visibility:visible;mso-wrap-style:square'>
 +
<v:imagedata src="Pumpsnotebook_files/image018.jpg" o:title="//lh6.googleusercontent.com/TQo0ALhzurvftLHK7JHJU21cHEF0B1SYa5t5HMs02gDfTiVbi9-PxpcCEqaaB5gAJh-_bK15m9Gm1Y5wcnJfmdt-afUAcNYNhVBR9IWiQcGw0w4eB7t_YHcwi5lDPT2bhgE"/>
 +
<v:textbox style='mso-rotate-with-shape:t'/>
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</v:shape><![endif]--><![if !vml]><img width=685 height=819
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src="Pumpsnotebook_files/image019.jpg"
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alt="https://lh6.googleusercontent.com/TQo0ALhzurvftLHK7JHJU21cHEF0B1SYa5t5HMs02gDfTiVbi9-PxpcCEqaaB5gAJh-_bK15m9Gm1Y5wcnJfmdt-afUAcNYNhVBR9IWiQcGw0w4eB7t_YHcwi5lDPT2bhgE"
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v:shapes="Picture_x0020_10"><![endif]></span><span style='font-size:10.0pt;
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font-family:Times;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
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 +
 +
<p class=MsoNormal><span style='font-size:16.0pt;font-family:Arial;mso-bidi-font-family:
 +
"Times New Roman";color:black'>Zymo research plasmid mini-prep from<i> E. coli</i>
 +
cultures</span><span style='font-size:10.0pt;font-family:Times;mso-bidi-font-family:
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Latest revision as of 03:34, 28 September 2013

Protocols




1. Make electrocompetent CB15N (for immediate use)

         0. Grow colony pick overnight CB15N in PYE @ 37Cº

1. Check OD600 of culture

2. Balance culture tubes < 0.01 g difference

3. Spin at 3000 rpm 4Cº for 15 minutes

4. Discard supernatant

5. Add 10 mL Milli Q water

6. Spin at 3000 rpm 4Cº for 15 minutes

7. Discard supernatant.

8. Repeat step 5-7 three times

           9. Make aliquots and put in deep freezer.



2. Miniprep on E. coli cultures

         1.Spin E. coli culture at 3000rpm for 5 minutes

         2. Discard supernatant.

         3. Resuspend in 6mL of Milli Q water.

         4. Add 1mL 6x Lysis buffer, invert 4-6 times, let sit for 5 minutes

         5. Add entire mix to blue zymo-midi-filter and  column, place  in 50mL conical tube. spin 5000rcf for 6 minutes

         6.Remove and discard filter and place column on collection tube. Place in benchtop centrifuge at max speed for 30 seconds.

         7. Add 400uL endo-wash and spin at max speed on benchtop centrifuge

         8. Add 400uL Zippy wash buffer , centrifuge at max speed for 1 minute, repeat once.

         9. Transfer to 1.5 mL Eppendorf tube add 150uL water, let sit for 1 minute before spinning at max speed for 1 minute

         10. Check concentration with nanodrop




        

3. Transfect CB15N with plasmids to make two strains of transformed CB15N.

4. Grow strains on plates.

5. Pick colonies for overnights.

6. Induce fluorescence.

7. Check for fluorescence.




Gentamicin Dilution



1. Weigh out .1g Gentamicin powder

2. Place in 50mL microcentrifuge tube

3. Add 10 mL Milli Q water

4. Pull plunger out of 60mL BD syringe

5. Screw on filter tip.

6. Fill syringe with solution

7. Empty syringe into new 50mL microcentifuge tube.



Sterilized colony pick

0. Autoclave growth media in container. (allow liberal space in container for aeration of culture), after cooling, add appropriate amount of antibiotic.

1. Heat tungsten loop on bunsen burner until red hot.

2. Let cool beside flame

3. Dip loop in agarose to check temperature

4. Beneath flame, carefully scrape-off one colony from plate making sure not to get any other colonies on loop.

5. Beneath flame, stick loop with colony in growth media making sure not to touch the sides of the container with loop. swirl around until colony washes off of loop.

6. Incubate in appropriate conditions.






Week 1

8-1-13

  • Made and autoclaved PYE
  • Prepared another batch of  electrocompetent caulobacter (culture OD600 = 0.615A)
  • Induced KAN resistant with xylose transformed caulobacter @ OD600 = 0.664A
  • Non induced : 3.25ml PXYFPC-2 + CB15N, 20.5ul antibiotic, 200ml PYE
  • Induced: 3.25ml PXYFPC-2+ CB15N, 20.5 ul antibiotic, 200ml PYE, .4g xylose .
  • Electroporation of CB15N with PVCFPC-4 .
  • Plated on PYE+agarose+gent plates 5 plates (10ul drop, 50ul drop, 100ul drop, two negative controls (see 8-5-13))
  • Plated CB15N on KAN plate for negative control. (note : this was not prepared following the electroporation protocol with water instead of plasmid. This was just a drop from a colony pick shaking incubator pick of CB15N )

8-2-13

  • Checked fluorescence of PCXFPC-2 : UI (OD600= .76 flouereced, I OD600 = .72 no fluorescence.)







Week 2

8-5-13

  • Checked plates
  • Negative control (CB15N+KAN electroporated with water): one colony
  • Negative control (CB15N+KAN straight from culture): many colonies
  • 10uL, 50uL, and 100uL many colonies
  • Result: Plates deemed unusable
  • Made new PYE + Gentamicin plates



8-6-13

  • PXYFPC-2 +CB15N culture to be induced made

         -5mL PXYFPC-2 + CB15N (OD600 = 0.72)

    • -20uL KAN
  • 50 uL PCR of  stpx and pflI on CB15N
  • Gel Electrophoresis on PCR reactions



8-7-13

  • Transformation of CB15N with PCXFPC-2 (electroporation) time constant = 5.6
  • Transformation of CB15N with PVCFPC-4 (electroporation) time constant = 5.4
  • PXYFPC-2 +CB15N culture to be induced made

         -5ml PXYFPC-2 + CB15N (OD600 = 0.72)

         -20ul KAN

·       TD PCR for Stpx and pflI tags from caulobacter, two 15 cycle sets

·       Initial denature of 95 Cº



denature

anneal

elongation

denature

anneal

elongation

95 Cº

56 Cº

68 Cº

95 Cº

49 Cº

68 Cº

:10

:15

2:20

:10

:15

2:20





Week 3

8-13-13

  • TD PCR for Stpx and pflI #3, 4, and 5. Two sets of 15 cycles with initial denature at 95 C for 1:00 for #3 and 94 C for 3:00 for #4 and #5



#3

denature

anneal

elongation

denature

anneal

elongation

95 Cº

56 Cº

68 Cº

95 Cº

49 Cº

68 Cº

:10

:15

2:20

:15

:15

2:20



#4

denature

anneal

elongation

denature

anneal

elongation

94 Cº

56 Cº

68 Cº

94 Cº

49 Cº

68 Cº

:15

:30

2:20

:15

:30

2:20



#5

denature

anneal

elongation

denature

anneal

elongation

94 Cº

59 Cº

68 Cº

94 Cº

52 Cº

68 Cº

:15

:15

2:30

:15

:15

2:30




8-15-13

  • Made LB
  • Checked plates

8-19-13

Gentamicin dilution (10mg/mL)

1. Weigh out 0.1g Gentamicin powder

2. Place in 50mL microcentrifuge tube

3. Add 10 mL Milli q water

4. Pull plunger out of 60mL BD syringe

5. Screw on filter tip

6. Fill syringe with solution

7. Empty syringe into new 50mL microcentrifuge tube.

8.aliquot 100uL, 10uL, 500uL, and 10uL into 1.5ml Eppendorf tubes



  • Sterilized inoculation of LB with E. coli + PXYFPC-2 +KAN (30ug/mL final concentration)
  • Sterilized inoculation of LB with E. coli + PVCFPC-4 +GENT (15ug/mL final concentration)



8-20-13

Created Glycerol stock of E. coli cultures containing PVCFPC-4

Plasmid Mini-prep of E. coli cultures

·       Culture #1 OD550 .916

·       Culture #2 OD5050 2.149

Nucleic Acid Concentration

A260

A280

260/280

260/230

Culture #1

21.0 ng/uL

.419

.221

1.89

1.68

Culture #2

299.2 ng/uL

5.983

3.184

1.88

2.13



Week 5

9-?-13

  • Use Geneious to design primers (forward and reverse) to get Halorhodopsin (HsHR) out of H.Salinarum.
  • Order primers.

9-3-13

  • Take chip of frozen glycerol stock H.Salinarum and suspend in Milli Q Water

9-4-13

25 uL PCR using HsHR primers ,HS (template), and GC master mix.

  • Negative control without primers labeled (-1)
  • Negative control without HS (template) labeled (-2)
  • 98Cº -2mins/98Cº 10s 57.2Cº 72Cº 2 min/72Cº 3 min hold  4Cº





9-5-13

  • Sterilized inoculation of SOC  with iGEM part BBa_K9000 “9000” + E. coli
  • Sterilized inoculation of SOC with iGEM part BBa_K9001 “9001” + E. coli
  • Sterilized inoculation of SOC with iGEM part BBa_K9010 “9010” + E. coli
  • Made chloramphenicol (1ug/1mL) plates



PCR of Halorhodopsin from Halobacterium Salinarum 30 cycles

25 uL reaction with 2x Phusion GC Master Mix



initial denature

denature

anneal

extension

final extension

98 Cº

98 Cº

57 Cº

72 Cº

72 Cº

2:00

00:10

00:20

1:00

5:00


https://lh5.googleusercontent.com/zoqmY8_jwDO6H4kdmXWKQDbayg_t_7tTOV2xuIHnLhfuI6g5P6IAC4aixAv79aV4FzsEX_GDs2rTSGPB9YVESOBxO_10EBqNa3XHbhmCxGzjC2mL5S14MKY_-g2gULexjho

9-6-13

Plasmid miniprep on 9000, 9010, and 9010 cultures.

         Results:

                  -9000 = 99.2 ng/ml

                  -9001 =99.7 ng/ml

                  -9010 = 38.1 ng/ml

         Gel legend : 1Kb ladder , 9010, 9001, 9000

  • Design and order sequencing primers for iGEM parts. same forward and reverse for all three.



Week 6

9-9-13

  • Send plasmids with sequencing primers out for sequencing.
  • Design and order primers for plasmid backbone.
  • 20uL PCR on HS for HsHR
  • Run diagnostic gel

         - Gel legend : 1Kb ladder , H? , +, -1, -2, https://lh5.googleusercontent.com/0Q85u88g6Sx3Xg9d2cGCLZjT4g7NqocTnSygFFPr4x_linJjMLnbRbHAI205lmA6Xj8KPv1-SCKYuEaqDhQxhe3hXULLqb4k1cWU7cLtNqLUiWHi1Q7nLKW58hr10V6UVsM



















9-10-13

  • 50 ul HsHR PCR labeled “50”
  • 98C 2min/ 98Cº :10s/57.2Cº 72Cº 1min/72min/Hold 4Cº



https://lh5.googleusercontent.com/z0GTplt-8fYo_yphibg41YCiunGWerxVFXSEPXuFcTzv9dRwAsJka3syaRQ6oM_koPX44Tejz8JeQvTLEbnaX2aCEQh0mOmYbmQ4D-drnfb-uudDOYdeSeUJQdltHoqZjNk












9-11-13

Purification gel

         -Gel Legend: 1Kb ladder, 50https://lh6.googleusercontent.com/kZa_3xX536Vv51c7eTVUpKXaR6GQmOhwebCL2oxD8a3m68bfWNXnZGquK-EY-x9CmYC4UUaajzn6qJPtGWfkKRh5WD-KHChocRyNonwjwdh7y_9RrlKDY68eHNND1Gf1zRk




















  • 50ul HsHR PCR (two reactions “50(1)” and “50(2)”) one negative control with no template “50 -”

Gel Legend : 1Kb ladder , 50(1), 50(2), 50-https://lh3.googleusercontent.com/zKPXvbXlR8t8qYT3XrTzaQkip94KqBpkA8Y4Cdflat05xNktHf1q-RY5GRdGIiB20p7SwrDf0V0cLxhjREvwIlVZ_h_19M-JuvjjtWwLoOR-JHBInZhkqeEcX321Qwd3SYY



9-12-13

50uL PCR to obtain plasmid backbone using 9010 (30.4ng/ul) as template.



initial denature

denature

anneal

extension

final extension

98 Cº

98 Cº

52.2 Cº

72 Cº

72 Cº

:30

:10

:30

1:00

5:00












Gel Legend : 1Kb ladder , 50(1), 50(2), 50-https://lh4.googleusercontent.com/LraY7dEsn1ebWvlutcCKsiU9hRs7QVvUkVoi7SedZHzyHJm1j0gWpDeYX3E7mYUY2UhKTL3t9dvCI-IOgIcxvTz9FWQQBr2iIHIs7jykiD0QyCtjvLXO31xbJxwWUsZ58fo



Two 20uL PCR reactions using part 9010 with different annealing temperatures. One at 55 Cº and one at 57 Cº















Gel Legend : 1Kb ladder , 9010(55 Cº) , 9010 neg (55 Cº), 9010 (57 Cº), 9010 neg (57Cº)


https://lh3.googleusercontent.com/5K0qLR04DEe-T0RhHNMtSAFiqwcXA7GKVEWPKcMez4AvAJkSmL1mP_0C6c84miYtFQFl4iy4y2qNe0EXgmQpTfz4eklCrLgmCc3eSES065g2q0E24XjDi_FghZp-eusnbiM




















9-14-13

20uL PCR to obtain plasmid backbone using linearized backbone as template.



Gel Legend : 1Kb ladder , 9010 57 C , 9010 57 C(-),  linear 57, linear 57 neg https://lh3.googleusercontent.com/Tsl3MNPOOovCvXGUArAeLgUvmGzZIqgHq0tkelEfgozwCS50xtEk-w9WaLuvW_mw-LA7MCNzxXrLJT8ErcyEsBwv2Ez2unP6-C5QxlcKnbUYH8Lo3qVvp3yVRgHsrEt9LLA









Week 7

9-15-13

  • New PCR of 9010 at 55 Cº and 57 Cº with new GC phusion mastermix due to contamination
  • a PCR of Linear PSB1C3 at 57 Cº



gel legend : 9010(55+), 9010(55 C-) , 9010(57 C+), 9010(57 C-), Linear (1), Linear (2), Linear(-), DNA ladder



9-16-13

Gel purification on PCR with linearized backbone



·       Run two samples from the PCR reaction, one with 5uL pcr and loading buffer with Sybr gold, and the other with the rest of the PCR (45uL) and loading buffer without Sybr gold.

·       Load the two samples in side by side lanes

·       Use the Sybr gold in uv light to find the band to cut out of the gel in the next lane over.

·       Centrifuge gel cut out with gel filter to separate. Collect filtrate.

·       Use Zymo research DNA Clean and Concentrate to recover DNA from filtrate



9-17-13

Concentrate DNA

1. Combine all gel purification tubes with purified DNA (add alcohol?)

2. Place separate cap with holes in it on top of tube.

3. Place tube in brick under vacuum chamber.

4. When all liquid is gone, add 5uL of Milli Q water.



9-18-13

Gibson reaction with KR2 G-blocks

         -5uL concentrated plasmid backbone

         -1uL of each G-block

         -3uL H2O

         -10uL 2x Gibson Master Mix

         -PCR Machine 50Cº for 1 hr.

Chemi-competent transformation of E.coli with Gibson reaction products.

  • Plate transformed E.coli on LB+Chloramphenicol plates. (400uL, 200uL, 100uL, and one with untransformed E. coli)




9-19-13

Suspend colonies in Milli Q water

  • Milli Q water with colonies colony PCR on colonies from 400uL and 200uL plates
  • 15 cycles

- 94 Cº initial denature 5 minutes

- 94 Cº denature 15 s

- 55 Cº first anneal 30s

- 68 Cº extension 1:00



  • 15 cycles anneal drop down 7 Cº

- 94 Cº denature :15

- 48 Cº second anneal  :30

- 68 Cº extension 1:00

- 68 Cº final extension 5:00

- 4 Cº hold infinite



Inoculation of E. coli colonies in LB + chloramphenicol




9-20-13



Run gel on PCR products from 9-19-13

Gel Legend: 1Kb ladder, “400”, “200”, negative control (no template)https://lh6.googleusercontent.com/TQo0ALhzurvftLHK7JHJU21cHEF0B1SYa5t5HMs02gDfTiVbi9-PxpcCEqaaB5gAJh-_bK15m9Gm1Y5wcnJfmdt-afUAcNYNhVBR9IWiQcGw0w4eB7t_YHcwi5lDPT2bhgE



Zymo research plasmid mini-prep from E. coli cultures







































 

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