Team:UC-Santa Cruz/Project/Overview

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pumps protocols (+ caulobacter expression protocols).docx

1. colony picks.

1.label two microcentrifuge tubes one with “E.Coli+PVCFPC-4+LB+GENT” and the other with “E.Coli +PXYFPC-2 +LB+KAN ” and make sure to put the date on each tube.

2. pour 30ml LB into tube labeled “E.Coli+PVCFPC-4+LB+GENT” add Gentamicin to final concentration of 15ug/ml.

3. pour 30ml LB into tube labeled “E.Coli+PXYFPC-2+LB+KAN” add Kanamycin to final concentration of 30ug/ml.

4.take p10 tip , scrape off ONE COLONY off of the plate that reads “LS4260 PXYFPC-2”(in fridge box that says iGEM on it), eject tip into into its respective microcentrifuge tube tube.

5. repeat with plate that reads “ LS4342 PVCFPC-4”

6. grow overnight at 37C.

2. make electrocompetent CB15N?

0. grow colony pick overnight CB15N in PYE @ 37C

1.7? check OD600 of culture

2?balence culture tubes < 0.01 g difference

3? spin at 3000 rpm 4C for 15 minutes

4? discard supernatant

5?add 10 ml Milli Q water

6? spin at 3000 rpm 4C for 15 minutes

7?discard supernatant

8?repeat step 6 and 7 3 times

9.? make aliquots and put in deep freezer.

3.miniprep on ecoli cultures

1.spin ecoli culture at 3000rpm for 5 minutes

2.discard supernatant.

3. resuspend in 6ml of Mili 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 5000 rcf 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 , centifuge at max speed for 1 minute, repeat once.

9. transfer to 1.5 ml epedorf 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.

Gentamycin Dilution

1.weigh out .1g Genamycin 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.

making LB aggar plates (one liter)

1. weight out 150 grams of aggar and add water for LB of 150 mg/ml

2.autoclave

3.cool to ~50C

5.add antibiotic

6.pour on plates

7.leave lid of plates slightly off to reduce condensation and contamination.

8.once plates have solidified, stack them with gel facing up

9. place in plastic bag to reduce drying out.

plasmid miniprep

materials

-zippy plasmid miniprep kit

-50 ml conical tubes

-1.5ml eppendorf tubes

-mili Q water

1. spin down cultures from 9-5-13

2. discard supernatant

3.add 6ml of mili q water

4. resuspend pellets by vortexing

5.add 1.5 ml 7x lysis buffer, invert tube 4-6 tubes, sit for 5 minutes.

6.add 3.5 ml of cold Neutralization Buffer (yellow), invert 4-6 times after yellow precipitate starts to form, invert 2-3 times.

7. place zymo-midi filter/zymo-spin-V-E column assembly into a clean 50 ml conical tube.

8.add 400ul of endo-wash buffer and centrifuge at 11,000 for 1 minute . Repeat for 1 minute to eliminate residue ( just column without filter)

9.transfer column into 1.5 ml microcentifuge tube and then add 150ul zippy elution buffer centrifuge for 1 minute after incubation for 1 minute.

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 contols (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 flourecece.)

Week 2

8-5-13

checked plates

neg control (CB15N+KAN electroporated with water): one colony

neg control (CB15N+KAN straight from culture): many colonies

10ul, 50ul, and 100ul many colonies

plates deemed unusable

Marshall Porter 8-5-13

Made new PYE + Gentamycin 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 electropheresis 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

Marshall Porter 8-7-13

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

● initial denature of 95 C

denature	anneal	elongation	denature	anneal	elongation
95 C	56 C	68 C	95 C	49 C	68 C
:10	:15	2:20	:10	:15	2:20

Gel?...unsucessful

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 C	56 C	68 C	95 C	49 C	68 C
:10	:15	2:20	:15	:15	2:20

#4

denature	anneal	elongation	denature	anneal	elongation
94 C	56 C	68 C	94 C	49 C	68 C
:15	:30	2:20	:15	:30	2:20

#5

denature	anneal	elongation	denature	anneal	elongation
94 C	59 C	68 C	94 C	52 C	68 C
: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 .1g genamycin powder

2.place in 50ml microcentrifuge tube

3.add 10 ml mili 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 ependorf tubes

sterilized enoculation of LB with ecoli + PXYFPC-2 +KAN (30ug/ml final concentration)

sterilized enocultion of LB with ecoli + PVCFPC-4 +GENT (15ug/ml final concentration)

8-20-13

Glycerol stock of E. coli cultures containing PVCFPC-4

Plasmid Midi-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 genious to design primers (forward and reverse) to get Halorhodopsin (HsHR) out of H.Salinuarium.

order primers.

9-3-13

take chip of frozen glycerol stock H.Salinarium and thaw in ?

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)

98C -2mins/98C 10s 57.2C 72C 2 min/72C 3min hold 4C

results gel?

9-5-13

sterilized inoculation of SOC with iGEM part BBa_K9000 “9000” +ecoli

sterilized enoculation of SOC with iGEM part BBa_K9001 “9001” +ecoli

sterilized enoculation of SOC with iGEM part BBa_K9010 “9010” +ecoli

made chloramphenicol (1ul/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 C	98 C	57 C	72 C	72 C
2:00	00:10	00:20	1:00	5:00

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 of parts

gell 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,

9-10-13

50 ul HsHR PCR labeled “50”

98C 2min/ 98C :10s/57.2C 72 1min/72min/Hold 4C

9-11-13

purification gel

-gel legend: 1Kb ladder, 50

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-

9-12-13

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

initial denature	denature	anneal	extension	final extension
98 C	98 C	52.2 C	72 C	72 C
:30	:10	:30	1:00	5:00

gel legend : 1Kb ladder , 50(1), 50(2), 50-

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

gel legend : 1Kb ladder , 9010(55 C) , 9010 neg (55 C), 9010 (57 C), 9010 neg (57C)

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

Week 7

9-15-13

New PCR of 9010 at 55 C and 57 C with new GC phusion mastermix due to contamination

and a PCR of Linear PSB1C3 at 57 C

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 sepreate 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 50C for 1 hr.

chemicompetent transformation of e.coli with Gibson reaction products.

plate transformed e.coli on LB+Chloramphenicol plates. (400ul , 200ul, 100ul, and one with untransformed ecoli)

9-19-13

suspend colonies in mili q water

mili q water with colonies colony PCR on colonies from 400ul and 200ul plates

15 cycles

94 C initial denature 5 minutes

94 C denature 15 s

55 C first anneal 30s

68 C extension 1:00

15 cycles anneal drop down 7 C

94 C denature :15

48 C second anneal :30

68 C extension 1:00

68 C final extension 5:00

4 C 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)

Zymo research plasmid midi-prep from e. coli cultures

9-21-13

Send psb1c3+KR2 plasmid in for sequencing.

9-24-13

receive sequencing results (not sure how to explain the results)

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+	margin-bottom:.0001pt;
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+	font-family:"Trebuchet MS";
+	mso-fareast-font-family:"Trebuchet MS";
+	mso-bidi-font-family:"Trebuchet MS";
+	color:#666666;}
+h4
+	{mso-style-parent:normal;
+	mso-style-next:normal;
+	margin-top:8.0pt;
+	margin-right:0in;
+	margin-bottom:0in;
+	margin-left:0in;
+	margin-bottom:.0001pt;
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+	line-height:115%;
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+	mso-bidi-font-family:"Trebuchet MS";
+	color:#666666;
+	font-weight:normal;
+	text-decoration:underline;
+	text-underline:single;}
+h4.CxSpFirst
+	{mso-style-parent:normal;
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+	mso-bidi-font-family:"Trebuchet MS";
+	color:#666666;
+	font-weight:normal;
+	text-decoration:underline;
+	text-underline:single;}
+h4.CxSpMiddle
+	{mso-style-parent:normal;
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+	mso-style-type:export-only;
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+	mso-bidi-font-family:"Trebuchet MS";
+	color:#666666;
+	font-weight:normal;
+	text-decoration:underline;
+	text-underline:single;}
+h4.CxSpLast
+	{mso-style-parent:normal;
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+	mso-style-type:export-only;
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+	mso-bidi-font-family:"Trebuchet MS";
+	color:#666666;
+	font-weight:normal;
+	text-decoration:underline;
+	text-underline:single;}
+h5
+	{mso-style-parent:normal;
+	mso-style-next:normal;
+	margin-top:8.0pt;
+	margin-right:0in;
+	margin-bottom:0in;
+	margin-left:0in;
+	margin-bottom:.0001pt;
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+	mso-bidi-font-size:12.0pt;
+	font-family:"Trebuchet MS";
+	mso-fareast-font-family:"Trebuchet MS";
+	mso-bidi-font-family:"Trebuchet MS";
+	color:#666666;
+	font-weight:normal;}
+h5.CxSpFirst
+	{mso-style-parent:normal;
+	mso-style-next:normal;
+	mso-style-type:export-only;
+	margin-top:8.0pt;
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+	margin-bottom:0in;
+	margin-left:0in;
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+	mso-fareast-font-family:"Trebuchet MS";
+	mso-bidi-font-family:"Trebuchet MS";
+	color:#666666;
+	font-weight:normal;}
+h5.CxSpMiddle
+	{mso-style-parent:normal;
+	mso-style-next:normal;
+	mso-style-type:export-only;
+	margin:0in;
+	margin-bottom:.0001pt;
+	mso-add-space:auto;
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+	mso-fareast-font-family:"Trebuchet MS";
+	mso-bidi-font-family:"Trebuchet MS";
+	color:#666666;
+	font-weight:normal;}
+h5.CxSpLast
+	{mso-style-parent:normal;
+	mso-style-next:normal;
+	mso-style-type:export-only;
+	margin:0in;
+	margin-bottom:.0001pt;
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+	mso-fareast-font-family:"Trebuchet MS";
+	mso-bidi-font-family:"Trebuchet MS";
+	color:#666666;
+	font-weight:normal;}
+h6
+	{mso-style-parent:normal;
+	mso-style-next:normal;
+	margin-top:8.0pt;
+	margin-right:0in;
+	margin-bottom:0in;
+	margin-left:0in;
+	margin-bottom:.0001pt;
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+	mso-bidi-font-size:12.0pt;
+	font-family:"Trebuchet MS";
+	mso-fareast-font-family:"Trebuchet MS";
+	mso-bidi-font-family:"Trebuchet MS";
+	color:#666666;
+	font-weight:normal;
+	font-style:italic;
+	mso-bidi-font-style:normal;}
+h6.CxSpFirst
+	{mso-style-parent:normal;
+	mso-style-next:normal;
+	mso-style-type:export-only;
+	margin-top:8.0pt;
+	margin-right:0in;
+	margin-bottom:0in;
+	margin-left:0in;
+	margin-bottom:.0001pt;
+	mso-add-space:auto;
+	line-height:115%;
+	mso-pagination:widow-orphan;
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+	mso-bidi-font-size:12.0pt;
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+	mso-fareast-font-family:"Trebuchet MS";
+	mso-bidi-font-family:"Trebuchet MS";
+	color:#666666;
+	font-weight:normal;
+	font-style:italic;
+	mso-bidi-font-style:normal;}
+h6.CxSpMiddle
+	{mso-style-parent:normal;
+	mso-style-next:normal;
+	mso-style-type:export-only;
+	margin:0in;
+	margin-bottom:.0001pt;
+	mso-add-space:auto;
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+	color:#666666;
+	font-weight:normal;
+	font-style:italic;
+	mso-bidi-font-style:normal;}
+h6.CxSpLast
+	{mso-style-parent:normal;
+	mso-style-next:normal;
+	mso-style-type:export-only;
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+	margin-bottom:.0001pt;
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+	color:#666666;
+	font-weight:normal;
+	font-style:italic;
+	mso-bidi-font-style:normal;}
+p.MsoTitle, li.MsoTitle, div.MsoTitle
+	{mso-style-parent:normal;
+	mso-style-next:normal;
+	margin:0in;
+	margin-bottom:.0001pt;
+	mso-add-space:auto;
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+	font-family:"Trebuchet MS";
+	mso-fareast-font-family:"Trebuchet MS";
+	mso-bidi-font-family:"Trebuchet MS";
+	color:black;}
+p.MsoTitleCxSpFirst, li.MsoTitleCxSpFirst, div.MsoTitleCxSpFirst
+	{mso-style-parent:normal;
+	mso-style-next:normal;
+	mso-style-type:export-only;
+	margin:0in;
+	margin-bottom:.0001pt;
+	mso-add-space:auto;
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+	mso-fareast-font-family:"Trebuchet MS";
+	mso-bidi-font-family:"Trebuchet MS";
+	color:black;}
+p.MsoTitleCxSpMiddle, li.MsoTitleCxSpMiddle, div.MsoTitleCxSpMiddle
+	{mso-style-parent:normal;
+	mso-style-next:normal;
+	mso-style-type:export-only;
+	margin:0in;
+	margin-bottom:.0001pt;
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+	mso-bidi-font-family:"Trebuchet MS";
+	color:black;}
+p.MsoTitleCxSpLast, li.MsoTitleCxSpLast, div.MsoTitleCxSpLast
+	{mso-style-parent:normal;
+	mso-style-next:normal;
+	mso-style-type:export-only;
+	margin:0in;
+	margin-bottom:.0001pt;
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+	font-family:"Trebuchet MS";
+	mso-fareast-font-family:"Trebuchet MS";
+	mso-bidi-font-family:"Trebuchet MS";
+	color:black;}
+p.MsoSubtitle, li.MsoSubtitle, div.MsoSubtitle
+	{mso-style-parent:normal;
+	mso-style-next:normal;
+	margin-top:0in;
+	margin-right:0in;
+	margin-bottom:10.0pt;
+	margin-left:0in;
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+	mso-bidi-font-family:"Trebuchet MS";
+	color:#666666;
+	font-style:italic;
+	mso-bidi-font-style:normal;}
+p.MsoSubtitleCxSpFirst, li.MsoSubtitleCxSpFirst, div.MsoSubtitleCxSpFirst
+	{mso-style-parent:normal;
+	mso-style-next:normal;
+	mso-style-type:export-only;
+	margin:0in;
+	margin-bottom:.0001pt;
+	mso-add-space:auto;
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+	mso-pagination:widow-orphan;
+	font-size:13.0pt;
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+	font-family:"Trebuchet MS";
+	mso-fareast-font-family:"Trebuchet MS";
+	mso-bidi-font-family:"Trebuchet MS";
+	color:#666666;
+	font-style:italic;
+	mso-bidi-font-style:normal;}
+p.MsoSubtitleCxSpMiddle, li.MsoSubtitleCxSpMiddle, div.MsoSubtitleCxSpMiddle
+	{mso-style-parent:normal;
+	mso-style-next:normal;
+	mso-style-type:export-only;
+	margin:0in;
+	margin-bottom:.0001pt;
+	mso-add-space:auto;
+	line-height:115%;
+	mso-pagination:widow-orphan;
+	font-size:13.0pt;
+	mso-bidi-font-size:12.0pt;
+	font-family:"Trebuchet MS";
+	mso-fareast-font-family:"Trebuchet MS";
+	mso-bidi-font-family:"Trebuchet MS";
+	color:#666666;
+	font-style:italic;
+	mso-bidi-font-style:normal;}
+p.MsoSubtitleCxSpLast, li.MsoSubtitleCxSpLast, div.MsoSubtitleCxSpLast
+	{mso-style-parent:normal;
+	mso-style-next:normal;
+	mso-style-type:export-only;
+	margin-top:0in;
+	margin-right:0in;
+	margin-bottom:10.0pt;
+	margin-left:0in;
+	mso-add-space:auto;
+	line-height:115%;
+	mso-pagination:widow-orphan;
+	font-size:13.0pt;
+	mso-bidi-font-size:12.0pt;
+	font-family:"Trebuchet MS";
+	mso-fareast-font-family:"Trebuchet MS";
+	mso-bidi-font-family:"Trebuchet MS";
+	color:#666666;
+	font-style:italic;
+	mso-bidi-font-style:normal;}
+p.normal, li.normal, div.normal
+	{mso-style-name:normal;
+	mso-style-parent:"";
+	margin:0in;
+	margin-bottom:.0001pt;
+	line-height:115%;
+	mso-pagination:widow-orphan;
+	font-size:11.0pt;
+	mso-bidi-font-size:12.0pt;
+	font-family:Arial;
+	mso-fareast-font-family:Arial;
+	mso-bidi-font-family:Arial;
+	color:black;}
+@page Section1
+	{size:8.5in 11.0in;
+	margin:1.0in 1.0in 1.0in 1.0in;
+	mso-header-margin:.5in;
+	mso-footer-margin:.5in;
+	mso-paper-source:0;}
+div.Section1
+	{page:Section1;}
+ /* List Definitions */
+@list l0
+	{mso-list-id:757169041;
+	mso-list-template-ids:2023524136;}
+@list l0:level1
+	{mso-level-number-format:bullet;
+	mso-level-text:\25CF;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	text-indent:.25in;
+	text-decoration:none;
+	text-underline:none;}
+@list l0:level2
+	{mso-level-number-format:bullet;
+	mso-level-text:\25CB;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	text-indent:.75in;
+	text-decoration:none;
+	text-underline:none;}
+@list l0:level3
+	{mso-level-number-format:bullet;
+	mso-level-text:\25A0;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	text-indent:1.25in;
+	text-decoration:none;
+	text-underline:none;}
+@list l0:level4
+	{mso-level-number-format:bullet;
+	mso-level-text:\25CF;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	text-indent:1.75in;
+	text-decoration:none;
+	text-underline:none;}
+@list l0:level5
+	{mso-level-number-format:bullet;
+	mso-level-text:\25CB;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	text-indent:2.25in;
+	text-decoration:none;
+	text-underline:none;}
+@list l0:level6
+	{mso-level-number-format:bullet;
+	mso-level-text:\25A0;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	text-indent:2.75in;
+	text-decoration:none;
+	text-underline:none;}
+@list l0:level7
+	{mso-level-number-format:bullet;
+	mso-level-text:\25CF;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	text-indent:3.25in;
+	text-decoration:none;
+	text-underline:none;}
+@list l0:level8
+	{mso-level-number-format:bullet;
+	mso-level-text:\25CB;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	text-indent:3.75in;
+	text-decoration:none;
+	text-underline:none;}
+@list l0:level9
+	{mso-level-number-format:bullet;
+	mso-level-text:\25A0;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	text-indent:4.25in;
+	text-decoration:none;
+	text-underline:none;}
+@list l1
+	{mso-list-id:1041436591;
+	mso-list-template-ids:-1633627142;}
+@list l1:level1
+	{mso-level-number-format:bullet;
+	mso-level-text:\25CF;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	text-indent:.25in;
+	text-decoration:none;
+	text-underline:none;}
+@list l1:level2
+	{mso-level-number-format:bullet;
+	mso-level-text:\25CB;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	text-indent:.75in;
+	text-decoration:none;
+	text-underline:none;}
+@list l1:level3
+	{mso-level-number-format:bullet;
+	mso-level-text:\25A0;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	text-indent:1.25in;
+	text-decoration:none;
+	text-underline:none;}
+@list l1:level4
+	{mso-level-number-format:bullet;
+	mso-level-text:\25CF;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	text-indent:1.75in;
+	text-decoration:none;
+	text-underline:none;}
+@list l1:level5
+	{mso-level-number-format:bullet;
+	mso-level-text:\25CB;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	text-indent:2.25in;
+	text-decoration:none;
+	text-underline:none;}
+@list l1:level6
+	{mso-level-number-format:bullet;
+	mso-level-text:\25A0;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	text-indent:2.75in;
+	text-decoration:none;
+	text-underline:none;}
+@list l1:level7
+	{mso-level-number-format:bullet;
+	mso-level-text:\25CF;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	text-indent:3.25in;
+	text-decoration:none;
+	text-underline:none;}
+@list l1:level8
+	{mso-level-number-format:bullet;
+	mso-level-text:\25CB;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	text-indent:3.75in;
+	text-decoration:none;
+	text-underline:none;}
+@list l1:level9
+	{mso-level-number-format:bullet;
+	mso-level-text:\25A0;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	text-indent:4.25in;
+	text-decoration:none;
+	text-underline:none;}
+@list l2
+	{mso-list-id:1566256833;
+	mso-list-template-ids:-1555673234;}
+@list l2:level1
+	{mso-level-number-format:bullet;
+	mso-level-text:\25CF;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	text-indent:.25in;
+	text-decoration:none;
+	text-underline:none;}
+@list l2:level2
+	{mso-level-number-format:bullet;
+	mso-level-text:\25CB;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	text-indent:.75in;
+	text-decoration:none;
+	text-underline:none;}
+@list l2:level3
+	{mso-level-number-format:bullet;
+	mso-level-text:\25A0;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	text-indent:1.25in;
+	text-decoration:none;
+	text-underline:none;}
+@list l2:level4
+	{mso-level-number-format:bullet;
+	mso-level-text:\25CF;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	text-indent:1.75in;
+	text-decoration:none;
+	text-underline:none;}
+@list l2:level5
+	{mso-level-number-format:bullet;
+	mso-level-text:\25CB;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	text-indent:2.25in;
+	text-decoration:none;
+	text-underline:none;}
+@list l2:level6
+	{mso-level-number-format:bullet;
+	mso-level-text:\25A0;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	text-indent:2.75in;
+	text-decoration:none;
+	text-underline:none;}
+@list l2:level7
+	{mso-level-number-format:bullet;
+	mso-level-text:\25CF;
+	mso-level-tab-stop:none;
+	mso-level-number-position:left;
+	text-indent:3.25in;
+	text-decoration:none;
+	text-underline:none;}
+@list l2:level8
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+	{margin-bottom:0in;}
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+	{margin-bottom:0in;}
+-->
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+<!--[if gte mso 10]>
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+ /* Style Definitions */
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+	{mso-style-name:"Table Normal";
+	mso-tstyle-rowband-size:0;
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+	mso-ascii-theme-font:minor-latin;
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+</style>
+<![endif]--><!--[if gte mso 9]><xml>
+ <o:shapedefaults v:ext="edit" spidmax="2050"/>
+</xml><![endif]--><!--[if gte mso 9]><xml>
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+  <o:idmap v:ext="edit" data="1"/>
+ </o:shapelayout></xml><![endif]-->
+</head>
+<body bgcolor=white lang=EN-US style='tab-interval:.5in'>
+<div class=Section1>
+<p class=normal>1. colony picks.</p>
+<p class=normal style='margin-left:.5in'>1.label two microcentrifuge tubes one
+with “E.Coli+PVCFPC-4+LB+GENT” and the other with<span style="mso-spacerun:
+yes">&nbsp;&nbsp; </span>“E.Coli +PXYFPC-2 +LB+KAN ” and make sure to put the
+date on each tube.</p>
+<p class=normal style='margin-left:.5in'>2. pour 30ml LB into<span
+style="mso-spacerun: yes">&nbsp; </span>tube labeled “E.Coli+PVCFPC-4+LB+GENT”
+add Gentamicin to final concentration of 15ug/ml.</p>
+<p class=normal style='margin-left:.5in'>3. pour 30ml LB into<span
+style="mso-spacerun: yes">&nbsp; </span>tube labeled “E.Coli+PXYFPC-2+LB+KAN”
+add Kanamycin<span style="mso-spacerun: yes">&nbsp; </span>to final
+concentration of 30ug/ml.</p>
+<p class=normal style='margin-left:.5in'>4.take p10 tip , scrape off ONE COLONY
+off of the plate that reads “LS4260<span style="mso-spacerun: yes">&nbsp;
+</span>PXYFPC-2”(in fridge box that says iGEM on it), eject tip into into its
+respective microcentrifuge tube tube.</p>
+<p class=normal style='text-indent:.5in'>5. repeat with plate that reads “
+LS4342 PVCFPC-4” </p>
+<p class=normal style='text-indent:.5in'>6. grow overnight at 37C.</p>
+<p class=normal style='text-indent:.5in'><o:p>&nbsp;</o:p></p>
+<p class=normal><span style="mso-spacerun: yes">&nbsp;</span></p>
+<p class=normal>2. make electrocompetent CB15N?</p>
+<p class=normal><span style="mso-spacerun: yes">&nbsp;</span><span
+style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>0.
+grow colony pick overnight CB15N in PYE @ 37C</p>
+<p class=normal style='text-indent:.5in'>1.7? check OD600 of culture</p>
+<p class=normal style='text-indent:.5in'>2?balence culture tubes &lt; 0.01 g
+difference</p>
+<p class=normal style='text-indent:.5in'>3? spin at 3000 rpm 4C for 15 minutes</p>
+<p class=normal style='text-indent:.5in'>4? discard supernatant</p>
+<p class=normal style='text-indent:.5in'>5?add 10 ml Milli Q water</p>
+<p class=normal style='text-indent:.5in'>6? spin at 3000 rpm 4C for 15 minutes</p>
+<p class=normal style='text-indent:.5in'>7?discard supernatant</p>
+<p class=normal style='text-indent:.5in'>8?repeat step 6 and 7 3 times</p>
+<p class=normal style='text-indent:.5in'>9.? make aliquots and put in deep
+freezer.</p>
+<p class=normal>3.miniprep on ecoli cultures</p>
+<p class=normal><span style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>1.spin
+ecoli culture at 3000rpm for 5 minutes</p>
+<p class=normal><span style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>2.discard
+supernatant.</p>
+<p class=normal><span style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>3.
+resuspend in 6ml of Mili Q water.</p>
+<p class=normal><span style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>4.add
+ml 6x Lysis buffer, invert 4-6 times, let sit for 5 minutes</p>
+<p class=normal style='text-align:justify;text-justify:inter-ideograph'><span
+style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>5.add
+entire mix to blue zymo-midi-filter and<span style="mso-spacerun: yes">&nbsp;
+</span>column, place<span style="mso-spacerun: yes">&nbsp; </span>in 50ml
+conical tube. spin 5000 rcf for 6 minutes</p>
+<p class=normal><span style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>6.remove
+and discard filter and place column on collection tube. Place in benchtop
+centrifuge at max speed for 30 seconds.</p>
+<p class=normal><span style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>7.add
+ul endo-wash and spin at max speed on benchtop centrifuge</p>
+<p class=normal><span style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>8.add
+ul zippy wash buffer , centifuge at max speed for 1 minute, repeat once.</p>
+<p class=normal><span style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>9.
+transfer to 1.5 ml epedorf tube add 150ul water, let sit for 1 minute before
+spinning at max speed for 1 minute</p>
+<p class=normal>10. check concentration with nanodrop </p>
+<p class=normal style='text-indent:.5in'><o:p>&nbsp;</o:p></p>
+<p class=normal style='text-indent:.5in'><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></p>
+<p class=normal>3.transfect CB15N with plasmids to make two strains of
+transformed CB15N.</p>
+<p class=normal>4.grow strains on plates.</p>
+<p class=normal>5.pick colonies for overnights.</p>
+<p class=normal>6.induce fluorescence.</p>
+<p class=normal>7.check for fluorescence. </p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal>Gentamycin Dilution</p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal>1.weigh out .1g Genamycin powder</p>
+<p class=normal>2.place in 50ml microcentrifuge tube</p>
+<p class=normal>3.add 10 ml milli q water</p>
+<p class=normal>4.pull plunger out of 60ml BD syringe</p>
+<p class=normal>5.screw on filter tip.</p>
+<p class=normal>6.fill syringe with solution</p>
+<p class=normal>7.empty syringe into new 50ml microcentifuge tube.</p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal>Sterilized colony pick </p>
+<p class=normal>0. autoclave growth media in container. (allow liberal space in
+container for aeration of culture), after cooling, add appropriate amount of
+antibiotic.</p>
+<p class=normal>1.heat tungsten loop on bunsen burner until red hot.</p>
+<p class=normal>2.let cool beside flame</p>
+<p class=normal>3.dip loop in agarose to check temperature</p>
+<p class=normal>4. beneath flame, carefully scrape-off one colony from plate
+making sure not to get any other colonies on loop.</p>
+<p class=normal>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.</p>
+<p class=normal>6.incubate in appropriate conditions.</p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal>making LB aggar plates (one liter)</p>
+<p class=normal>1. weight out 150 grams of aggar and add water for LB of 150
+mg/ml</p>
+<p class=normal>2.autoclave</p>
+<p class=normal>3.cool to ~50C</p>
+<p class=normal>5.add antibiotic</p>
+<p class=normal>6.pour on plates</p>
+<p class=normal>7.leave lid of plates slightly off to reduce condensation and
+contamination.</p>
+<p class=normal>8.once plates have solidified, stack them with gel facing up</p>
+<p class=normal>9. place in plastic bag to reduce drying out.</p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal>plasmid miniprep</p>
+<p class=normal><span style="mso-spacerun: yes">&nbsp;</span>materials</p>
+<p class=normal><span style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>-zippy
+plasmid miniprep kit</p>
+<p class=normal><span style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>-50
+ml conical tubes</p>
+<p class=normal><span style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>-1.5ml
+eppendorf tubes</p>
+<p class=normal><span style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>-mili
+Q water</p>
+<p class=normal>1. spin down cultures from 9-5-13</p>
+<p class=normal>2. discard supernatant</p>
+<p class=normal>3.add 6ml of mili q water</p>
+<p class=normal>4. resuspend pellets by vortexing</p>
+<p class=normal>5.add 1.5 ml 7x lysis buffer, invert tube 4-6 tubes, sit for 5
+minutes.</p>
+<p class=normal>6.add 3.5 ml of cold Neutralization Buffer (yellow), invert 4-6
+times after yellow precipitate starts to form, invert 2-3 times.</p>
+<p class=normal>7. place zymo-midi filter/zymo-spin-V-E column assembly into a
+clean 50 ml conical tube.</p>
+<p class=normal>8.add 400ul of endo-wash buffer and centrifuge at 11,000 for 1
+minute . Repeat for 1 minute to eliminate residue ( just column without filter)</p>
+<p class=normal>9.transfer column into 1.5 ml microcentifuge tube and then add
+ul zippy elution buffer centrifuge for 1 minute after incubation for 1
+minute.</p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><u><span style='font-size:24.0pt;mso-bidi-font-size:12.0pt;
+line-height:115%;color:red'>Week 1</span></u></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>8-1-13</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>Made and
+autoclaved PYE</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>prepared
+another batch of<span style="mso-spacerun: yes">&nbsp; </span>electrocompetent
+caulobacter (culture OD600 = 0.615A)</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>induced KAN
+resistant with xylose transformed caulobacter @ OD600 = 0.664A </span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>non induced :
+.25ml </span>PXYFPC-2<span style='font-size:12.0pt;line-height:115%'> + CB15N,
+.5ul antibiotic, 200ml PYE</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>induced: 3.25ml
+</span>PXYFPC-2<span style='font-size:12.0pt;line-height:115%'>+ CB15N, 20.5 ul
+antibiotic, 200ml PYE, .4g xylose .</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>electroporation
+of CB15N with </span>PVCFPC-4<span style='font-size:12.0pt;line-height:115%'> .</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>plated on
+PYE+agarose+gent plates 5 plates (10ul drop, 50ul drop, 100ul drop, two
+negative contols (see 8-5-13))</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>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 )</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>8-2-13</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>checked fluorescence
+of PCXFPC-2 : UI (OD600= .76 flouereced, I OD600 = .72 no flourecece.)</span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><u><span style='font-size:24.0pt;mso-bidi-font-size:12.0pt;
+line-height:115%;color:red'>Week 2</span></u></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>8-5-13</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>checked plates </span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>neg control
+(CB15N+KAN electroporated with water): one colony</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>neg control
+(CB15N+KAN straight from culture): many colonies</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>10ul, 50ul, and
+ul many colonies</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>plates deemed
+unusable</span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>Marshall Porter
+<b style='mso-bidi-font-weight:normal'>8-5-13</b></span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>Made new PYE +
+Gentamycin plates</span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>8-6-13</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>PXYFPC-2 +CB15N
+culture to be induced made </span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'><span
+style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>-5ml
+PXYFPC-2 + CB15N (OD600 = 0.72)</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'><span
+style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>-20ul
+KAN</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>50 ul PCR
+of<span style="mso-spacerun: yes">&nbsp; </span>stpx and pflI on CB15N</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>gel
+electropheresis on PCR reactions </span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>8-7-13</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>transformation
+of CB15N with PCXFPC-2 (electroporation) time constant = 5.6</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>transformation
+of CB15N with </span>PVCFPC-4<span style='font-size:12.0pt;line-height:115%'>
+(electroporation) time constant = 5.4</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>PXYFPC-2 +CB15N
+culture to be induced made </span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'><span
+style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>-5ml
+PXYFPC-2 + CB15N (OD600 = 0.72)</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'><span
+style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>-20ul
+KAN</span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>Marshall Porter
+<b style='mso-bidi-font-weight:normal'>8-7-13</b></span></p>
+<p class=normalCxSpMiddle style='margin-left:.5in;mso-add-space:auto;
+text-indent:-17.95pt;mso-list:l2 level1 lfo3'><![if !supportLists]><span
+style='font-size:12.0pt;line-height:115%'><span style='mso-list:Ignore'>&#9679;<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:12.0pt;line-height:115%'>TD PCR for Stpx and pflI tags from
+caulobacter, two 15 cycle sets <o:p></o:p></span></p>
+<p class=normalCxSpMiddle style='margin-left:.5in;mso-add-space:auto;
+text-indent:-17.95pt;mso-list:l2 level1 lfo3'><![if !supportLists]><span
+style='font-size:12.0pt;line-height:115%'><span style='mso-list:Ignore'>&#9679;<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:12.0pt;line-height:115%'>initial denature of 95 C<o:p></o:p></span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0
+ style='margin-left:4.5pt;border-collapse:collapse;mso-table-layout-alt:fixed;
+ border:none;mso-border-alt:solid black 1.0pt;mso-yfti-tbllook:1184;mso-padding-alt:
+in .5pt 0in .5pt;mso-border-insideh:1.0pt solid black;mso-border-insidev:
+.0pt solid black'>
+ <tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>denature
+  </span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>anneal</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>elongation</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>denature</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>anneal</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>elongation</span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:1'>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>95
+  C</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>56
+  C</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>68
+  C</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>95
+  C</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>49
+  C</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>68
+  C</span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:2;mso-yfti-lastrow:yes'>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>:10
+  </span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>:15
+  </span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>2:20</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>:10</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>:15</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>2:20</span></p>
+  </td>
+ </tr>
+</table>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%;color:blue'>Gel?...unsucessful</span></p>
+<p class=normal><u><span style='font-size:24.0pt;mso-bidi-font-size:12.0pt;
+line-height:115%;color:red'>Week 3</span></u></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>8-13-13</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>TD PCR for Stpx
+and pflI #3, 4, and 5. Two sets of 15 cycles with initial denature at 95 C for
+:00 for #3 and 94 C for 3:00 for #4 and #5</span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>#3</span></p>
+<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0
+ style='margin-left:4.5pt;border-collapse:collapse;mso-table-layout-alt:fixed;
+ border:none;mso-border-alt:solid black 1.0pt;mso-yfti-tbllook:1184;mso-padding-alt:
+in .5pt 0in .5pt;mso-border-insideh:1.0pt solid black;mso-border-insidev:
+.0pt solid black'>
+ <tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>denature
+  </span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>anneal</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>elongation</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>denature</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>anneal</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>elongation</span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:1'>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>95
+  C</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>56
+  C</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>68
+  C</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>95
+  C</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>49
+  C</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>68
+  C</span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:2;mso-yfti-lastrow:yes'>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>:10
+  </span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>:15
+  </span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>2:20</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>:15</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>:15</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>2:20</span></p>
+  </td>
+ </tr>
+</table>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>#4</span></p>
+<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0
+ style='margin-left:4.5pt;border-collapse:collapse;mso-table-layout-alt:fixed;
+ border:none;mso-border-alt:solid black 1.0pt;mso-yfti-tbllook:1184;mso-padding-alt:
+in .5pt 0in .5pt;mso-border-insideh:1.0pt solid black;mso-border-insidev:
+.0pt solid black'>
+ <tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>denature
+  </span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>anneal</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>elongation</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>denature</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>anneal</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>elongation</span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:1'>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>94
+  C</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>56
+  C</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>68
+  C</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>94
+  C</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>49
+  C</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>68
+  C</span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:2;mso-yfti-lastrow:yes'>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>:15
+  </span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>:30
+  </span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>2:20</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>:15</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>:30</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>2:20</span></p>
+  </td>
+ </tr>
+</table>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>#5</span></p>
+<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0
+ style='margin-left:4.5pt;border-collapse:collapse;mso-table-layout-alt:fixed;
+ border:none;mso-border-alt:solid black 1.0pt;mso-yfti-tbllook:1184;mso-padding-alt:
+in .5pt 0in .5pt;mso-border-insideh:1.0pt solid black;mso-border-insidev:
+.0pt solid black'>
+ <tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>denature
+  </span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>anneal</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>elongation</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>denature</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>anneal</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>elongation</span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:1'>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>94
+  C</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>59
+  C</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>68
+  C</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>94
+  C</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>52
+  C</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>68
+  C</span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:2;mso-yfti-lastrow:yes'>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>:15
+  </span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>:15
+  </span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>2:30</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>:15</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>:15</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>2:30</span></p>
+  </td>
+ </tr>
+</table>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>8-15-13</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>made LB</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>checked plates?</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>8-19-13</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>Gentamicin
+dilution (10mg/ml)</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>1.weigh out .1g
+genamycin powder</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>2.place in 50ml
+microcentrifuge tube</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>3.add 10 ml
+mili q water</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>4.pull plunger
+out of 60ml BD syringe</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>5.screw on
+filter tip</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>6.fill syringe
+with solution </span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>7.empty syringe
+into new 50ml microcentrifuge tube.</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>8.aliquot
+ul, 10ul, 500ul, and 10ul into 1.5ml ependorf tubes</span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>sterilized
+enoculation of LB with ecoli + PXYFPC-2 +KAN (30ug/ml final concentration)</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>sterilized
+enocultion of LB with ecoli + PVCFPC-4 +GENT (15ug/ml final concentration)</span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>8-20-13</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>Glycerol stock
+of E. coli cultures containing PVCFPC-4</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>Plasmid
+Midi-prep of E. coli cultures</span></p>
+<p class=normalCxSpMiddle style='margin-left:.5in;mso-add-space:auto;
+text-indent:-17.95pt;mso-list:l0 level1 lfo1'><![if !supportLists]><span
+style='font-size:12.0pt;line-height:115%'><span style='mso-list:Ignore'>&#9679;<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:12.0pt;line-height:115%'>Culture #1 OD550 .916<o:p></o:p></span></p>
+<p class=normalCxSpMiddle style='margin-left:.5in;mso-add-space:auto;
+text-indent:-17.95pt;mso-list:l0 level1 lfo1'><![if !supportLists]><span
+style='font-size:12.0pt;line-height:115%'><span style='mso-list:Ignore'>&#9679;<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:12.0pt;line-height:115%'>Culture #2 OD5050 2.149<o:p></o:p></span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0
+ style='margin-left:4.5pt;border-collapse:collapse;mso-table-layout-alt:fixed;
+ border:none;mso-border-alt:solid black 1.0pt;mso-yfti-tbllook:1184;mso-padding-alt:
+in .5pt 0in .5pt;mso-border-insideh:1.0pt solid black;mso-border-insidev:
+.0pt solid black'>
+ <tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
+  <td width=98 valign=top style='width:98.25pt;border:solid black 1.0pt;
+  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><o:p>&nbsp;</o:p></p>
+  </td>
+  <td width=58 valign=top style='width:57.75pt;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>Nucleic
+  Acid Concentration</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>A260</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>A280</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>260/280</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>260/230</span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:1'>
+  <td width=98 valign=top style='width:98.25pt;border:solid black 1.0pt;
+  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>Culture
+  #1</span></p>
+  </td>
+  <td width=58 valign=top style='width:57.75pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>21.0
+  ng/ul</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>.419</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>.221</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>1.89</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>1.68</span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:2;mso-yfti-lastrow:yes'>
+  <td width=98 valign=top style='width:98.25pt;border:solid black 1.0pt;
+  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>Culture
+  #2</span></p>
+  </td>
+  <td width=58 valign=top style='width:57.75pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>299.2
+  ng/ul</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>5.983</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>3.184</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>1.88</span></p>
+  </td>
+  <td width=78 valign=top style='width:78.0pt;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>2.13</span></p>
+  </td>
+ </tr>
+</table>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><u><span style='font-size:24.0pt;mso-bidi-font-size:12.0pt;
+line-height:115%;color:red'>Week 5</span></u></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>9-?-13</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>use genious to
+design primers (forward and reverse) to get Halorhodopsin (HsHR) out of
+H.Salinuarium.</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>order primers.</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>9-3-13</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>take chip of
+frozen glycerol stock H.Salinarium and thaw in ?</span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>9-4-13</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>25 ul PCR using
+HsHR primers ,HS (template), and GC master mix.</span></p>
+<p class=normal style='text-indent:.5in'><span style='font-size:12.0pt;
+line-height:115%'>negative control without primers labeled (-1)</span></p>
+<p class=normal style='text-indent:.5in'><span style='font-size:12.0pt;
+line-height:115%'>negative control without HS (template) labeled (-2)</span></p>
+<p class=normal style='text-indent:.5in'><span style='font-size:12.0pt;
+line-height:115%'>98C -2mins/98C 10s 57.2C 72C 2 min/72C 3min hold<span
+style="mso-spacerun: yes">&nbsp; </span>4C</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>r</span><span
+style='font-size:12.0pt;line-height:115%;color:blue'>esults gel?</span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>9-5-13</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>sterilized
+inoculation of SOC<span style="mso-spacerun: yes">&nbsp; </span>with iGEM part
+BBa_K9000 “9000” +ecoli</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>sterilized
+enoculation of SOC with iGEM part BBa_K9001 “9001” +ecoli</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>sterilized
+enoculation of SOC with iGEM part BBa_K9010 “9010” +ecoli</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>made
+chloramphenicol (1ul/1ml) plates</span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>PCR of
+Halorhodopsin from Halobacterium Salinarum 30 cycles</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>25 ul reaction
+with 2x phusion GC Master Mix</span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0
+ style='margin-left:4.5pt;border-collapse:collapse;mso-table-layout-alt:fixed;
+ border:none;mso-border-alt:solid black 1.0pt;mso-yfti-tbllook:1184;mso-padding-alt:
+in .5pt 0in .5pt;mso-border-insideh:1.0pt solid black;mso-border-insidev:
+.0pt solid black'>
+ <tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
+  <td width=94 valign=top style='width:1.3in;border:solid black 1.0pt;
+  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>initial
+  denature</span></p>
+  </td>
+  <td width=94 valign=top style='width:1.3in;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>denature</span></p>
+  </td>
+  <td width=94 valign=top style='width:1.3in;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>anneal</span></p>
+  </td>
+  <td width=94 valign=top style='width:1.3in;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>extension</span></p>
+  </td>
+  <td width=94 valign=top style='width:1.3in;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>final
+  extension</span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:1'>
+  <td width=94 valign=top style='width:1.3in;border:solid black 1.0pt;
+  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>98
+  C</span></p>
+  </td>
+  <td width=94 valign=top style='width:1.3in;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>98
+  C</span></p>
+  </td>
+  <td width=94 valign=top style='width:1.3in;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>57
+  C</span></p>
+  </td>
+  <td width=94 valign=top style='width:1.3in;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>72
+  C</span></p>
+  </td>
+  <td width=94 valign=top style='width:1.3in;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>72
+  C</span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:2;mso-yfti-lastrow:yes'>
+  <td width=94 valign=top style='width:1.3in;border:solid black 1.0pt;
+  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>2:00</span></p>
+  </td>
+  <td width=94 valign=top style='width:1.3in;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>00:10
+  </span></p>
+  </td>
+  <td width=94 valign=top style='width:1.3in;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>00:20</span></p>
+  </td>
+  <td width=94 valign=top style='width:1.3in;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>1:00</span></p>
+  </td>
+  <td width=94 valign=top style='width:1.3in;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>5:00</span></p>
+  </td>
+ </tr>
+</table>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><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="image04.jpg" o:spid="_x0000_i1034" type="#_x0000_t75"
+ style='width:349pt;height:417pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="pumpsprotocolscaulobacterexpressionprotocols_files/image001.jpg"
+  o:title=""/>
+ <v:textbox style='mso-rotate-with-shape:t'/>
+</v:shape><![endif]--><![if !vml]><img width=351 height=419
+src="pumpsprotocolscaulobacterexpressionprotocols_files/image002.gif" v:shapes="image04.jpg"><![endif]></span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>9-6-13</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>plasmid
+miniprep on 9000, 9010, and 9010 cultures.</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'><span
+style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>results
+</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'><span
+style='mso-tab-count:2'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>-9000
+= 99.2 ng/ml</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'><span
+style='mso-tab-count:2'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>-9001
+=99.7 ng/ml</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'><span
+style='mso-tab-count:2'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>-9010
+= 38.1 ng/ml</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>gel of parts </span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'><span
+style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>gell
+legend : 1Kb ladder , 9010, 9001, 9000</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>design and
+order sequencing primers for iGEM parts. same forward and reverse for all
+three.</span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><u><span style='font-size:24.0pt;mso-bidi-font-size:12.0pt;
+line-height:115%;color:red'>Week 6</span></u></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>9-9-13</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>send plasmids
+with sequencing primers out for sequencing.</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>design and order
+primers for plasmid backbone.</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>20ul PCR on HS
+for HsHR</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>run diagnostic
+gel </span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'><span
+style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>-gel
+legend : 1Kb ladder , H? , +, -1, -2, </span></p>
+<p class=normal><span style='mso-no-proof:yes'><!--[if gte vml 1]><v:shape
+ id="image09.jpg" o:spid="_x0000_i1033" type="#_x0000_t75" style='width:469pt;
+ height:352pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="pumpsprotocolscaulobacterexpressionprotocols_files/image003.jpg"
+  o:title=""/>
+ <v:textbox style='mso-rotate-with-shape:t'/>
+</v:shape><![endif]--><![if !vml]><img width=471 height=354
+src="pumpsprotocolscaulobacterexpressionprotocols_files/image004.gif" v:shapes="image09.jpg"><![endif]></span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>9-10-13</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>50 ul HsHR PCR
+labeled “50”</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>98C 2min/ 98C
+:10s/57.2C 72 1min/72min/Hold 4C</span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='mso-no-proof:yes'><!--[if gte vml 1]><v:shape
+ id="image06.jpg" o:spid="_x0000_i1032" type="#_x0000_t75" style='width:4in;
+ height:348pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="pumpsprotocolscaulobacterexpressionprotocols_files/image005.jpg"
+  o:title=""/>
+ <v:textbox style='mso-rotate-with-shape:t'/>
+</v:shape><![endif]--><![if !vml]><img width=290 height=350
+src="pumpsprotocolscaulobacterexpressionprotocols_files/image006.gif" v:shapes="image06.jpg"><![endif]></span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>9-11-13</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>purification
+gel</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'><span
+style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>-gel
+legend: 1Kb ladder, 50</span></p>
+<p class=normal><span style='mso-no-proof:yes'><!--[if gte vml 1]><v:shape
+ id="image00.jpg" o:spid="_x0000_i1031" type="#_x0000_t75" style='width:251pt;
+ height:279pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="pumpsprotocolscaulobacterexpressionprotocols_files/image007.jpg"
+  o:title=""/>
+ <v:textbox style='mso-rotate-with-shape:t'/>
+</v:shape><![endif]--><![if !vml]><img width=253 height=281
+src="pumpsprotocolscaulobacterexpressionprotocols_files/image008.gif" v:shapes="image00.jpg"><![endif]></span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>50ul HsHR PCR
+(two reactions “50(1)” and “50(2)”) one negative control with no template “50
+-”</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>gel legend :
+Kb ladder , 50(1), 50(2), 50-</span></p>
+<p class=normal><span style='mso-no-proof:yes'><!--[if gte vml 1]><v:shape
+ id="image05.jpg" o:spid="_x0000_i1030" type="#_x0000_t75" style='width:218pt;
+ height:276pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="pumpsprotocolscaulobacterexpressionprotocols_files/image009.jpg"
+  o:title=""/>
+ <v:textbox style='mso-rotate-with-shape:t'/>
+</v:shape><![endif]--><![if !vml]><img width=220 height=278
+src="pumpsprotocolscaulobacterexpressionprotocols_files/image010.gif" v:shapes="image05.jpg"><![endif]></span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>9-12-13</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>50ul PCR to
+obtain plasmid backbone using 9010 (30.4ng/ul) as template. </span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0
+ style='margin-left:4.5pt;border-collapse:collapse;mso-table-layout-alt:fixed;
+ border:none;mso-border-alt:solid black 1.0pt;mso-yfti-tbllook:1184;mso-padding-alt:
+in .5pt 0in .5pt;mso-border-insideh:1.0pt solid black;mso-border-insidev:
+.0pt solid black'>
+ <tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
+  <td width=94 valign=top style='width:1.3in;border:solid black 1.0pt;
+  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>initial
+  denature</span></p>
+  </td>
+  <td width=94 valign=top style='width:1.3in;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>denature</span></p>
+  </td>
+  <td width=94 valign=top style='width:1.3in;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>anneal</span></p>
+  </td>
+  <td width=94 valign=top style='width:1.3in;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>extension</span></p>
+  </td>
+  <td width=94 valign=top style='width:1.3in;border:solid black 1.0pt;
+  border-left:none;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>final
+  extension</span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:1'>
+  <td width=94 valign=top style='width:1.3in;border:solid black 1.0pt;
+  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>98
+  C</span></p>
+  </td>
+  <td width=94 valign=top style='width:1.3in;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>98
+  C</span></p>
+  </td>
+  <td width=94 valign=top style='width:1.3in;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>52.2
+  C</span></p>
+  </td>
+  <td width=94 valign=top style='width:1.3in;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>72
+  C</span></p>
+  </td>
+  <td width=94 valign=top style='width:1.3in;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>72
+  C</span></p>
+  </td>
+ </tr>
+ <tr style='mso-yfti-irow:2;mso-yfti-lastrow:yes'>
+  <td width=94 valign=top style='width:1.3in;border:solid black 1.0pt;
+  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>:30</span></p>
+  </td>
+  <td width=94 valign=top style='width:1.3in;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>:10</span></p>
+  </td>
+  <td width=94 valign=top style='width:1.3in;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>:30</span></p>
+  </td>
+  <td width=94 valign=top style='width:1.3in;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>1:00</span></p>
+  </td>
+  <td width=94 valign=top style='width:1.3in;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:5.0pt 5.0pt 5.0pt 5.0pt'>
+  <p class=normal style='line-height:normal'><span style='font-size:12.0pt'>5:00</span></p>
+  </td>
+ </tr>
+</table>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>gel legend :
+Kb ladder , 50(1), 50(2), 50-</span></p>
+<p class=normal><span style='mso-no-proof:yes'><!--[if gte vml 1]><v:shape
+ id="image03.jpg" o:spid="_x0000_i1029" type="#_x0000_t75" style='width:295pt;
+ height:347pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="pumpsprotocolscaulobacterexpressionprotocols_files/image011.jpg"
+  o:title=""/>
+ <v:textbox style='mso-rotate-with-shape:t'/>
+</v:shape><![endif]--><![if !vml]><img width=297 height=349
+src="pumpsprotocolscaulobacterexpressionprotocols_files/image012.gif" v:shapes="image03.jpg"><![endif]></span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>Two 20ul PCR
+reactions using part 9010 with different annealing temperatures. One at 55 C
+and one at 57 C</span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>gel legend :
+Kb ladder , 9010(55 C) , 9010 neg (55 C), 9010 (57 C), 9010 neg (57C) </span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='mso-no-proof:yes'><!--[if gte vml 1]><v:shape
+ id="image02.jpg" o:spid="_x0000_i1028" type="#_x0000_t75" style='width:244pt;
+ height:278pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="pumpsprotocolscaulobacterexpressionprotocols_files/image013.jpg"
+  o:title=""/>
+ <v:textbox style='mso-rotate-with-shape:t'/>
+</v:shape><![endif]--><![if !vml]><img width=246 height=280
+src="pumpsprotocolscaulobacterexpressionprotocols_files/image014.gif" v:shapes="image02.jpg"><![endif]></span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>9-14-13</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>20ul PCR to
+obtain plasmid backbone using linearized backbone as template.</span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>gel legend :
+Kb ladder , 9010 57 C , 9010 57 C(-),<span style="mso-spacerun: yes">&nbsp;
+</span>linear 57, linear 57 neg </span></p>
+<p class=normal><span style='mso-no-proof:yes'><!--[if gte vml 1]><v:shape
+ id="image08.jpg" o:spid="_x0000_i1027" type="#_x0000_t75" style='width:311pt;
+ height:348pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="pumpsprotocolscaulobacterexpressionprotocols_files/image015.jpg"
+  o:title=""/>
+ <v:textbox style='mso-rotate-with-shape:t'/>
+</v:shape><![endif]--><![if !vml]><img width=313 height=350
+src="pumpsprotocolscaulobacterexpressionprotocols_files/image016.gif" v:shapes="image08.jpg"><![endif]></span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><u><span style='font-size:24.0pt;mso-bidi-font-size:12.0pt;
+line-height:115%;color:red'>Week 7</span></u></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>9-15-13</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>New PCR of 9010
+at 55 C and 57 C with new GC phusion mastermix due to contamination</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>and a PCR of
+Linear PSB1C3 at 57 C</span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>gel legend :
+(55+), 9010(55 C-) , 9010(57 C+), 9010(57 C-), Linear (1), Linear (2),
+Linear(-), DNA ladder</span></p>
+<p class=normal><span style='mso-no-proof:yes'><!--[if gte vml 1]><v:shape
+ id="image01.jpg" o:spid="_x0000_i1026" type="#_x0000_t75" style='width:375pt;
+ height:374pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="pumpsprotocolscaulobacterexpressionprotocols_files/image017.jpg"
+  o:title=""/>
+ <v:textbox style='mso-rotate-with-shape:t'/>
+</v:shape><![endif]--><![if !vml]><img width=377 height=376
+src="pumpsprotocolscaulobacterexpressionprotocols_files/image018.gif" v:shapes="image01.jpg"><![endif]></span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>9-16-13</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>Gel purification
+on PCR with linearized backbone </span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normalCxSpMiddle style='margin-left:.5in;mso-add-space:auto;
+text-indent:-17.95pt;mso-list:l1 level1 lfo2'><![if !supportLists]><span
+style='font-size:12.0pt;line-height:115%'><span style='mso-list:Ignore'>&#9679;<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:12.0pt;line-height:115%'>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=normalCxSpMiddle style='margin-left:.5in;mso-add-space:auto;
+text-indent:-17.95pt;mso-list:l1 level1 lfo2'><![if !supportLists]><span
+style='font-size:12.0pt;line-height:115%'><span style='mso-list:Ignore'>&#9679;<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:12.0pt;line-height:115%'>Load the two samples in side by side
+lanes<o:p></o:p></span></p>
+<p class=normalCxSpMiddle style='margin-left:.5in;mso-add-space:auto;
+text-indent:-17.95pt;mso-list:l1 level1 lfo2'><![if !supportLists]><span
+style='font-size:12.0pt;line-height:115%'><span style='mso-list:Ignore'>&#9679;<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:12.0pt;line-height:115%'>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=normalCxSpMiddle style='margin-left:.5in;mso-add-space:auto;
+text-indent:-17.95pt;mso-list:l1 level1 lfo2'><![if !supportLists]><span
+style='font-size:12.0pt;line-height:115%'><span style='mso-list:Ignore'>&#9679;<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:12.0pt;line-height:115%'>Centrifuge gel cut out with gel
+filter to separate. Collect filtrate.<o:p></o:p></span></p>
+<p class=normalCxSpMiddle style='margin-left:.5in;mso-add-space:auto;
+text-indent:-17.95pt;mso-list:l1 level1 lfo2'><![if !supportLists]><span
+style='font-size:12.0pt;line-height:115%'><span style='mso-list:Ignore'>&#9679;<span
+style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+style='font-size:12.0pt;line-height:115%'>Use Zymo research DNA Clean and
+Concentrate to recover DNA from filtrate<o:p></o:p></span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>9-17-13</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>concentrate DNA</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>1.combine all
+gel purification tubes with purified DNA (add alcohol?)</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>2.place
+sepreate cap with holes in it on top of tube.</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>3.place tube in
+brick under vacuum chamber.</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>4.when all
+liquid is gone, add 5ul of Milli Q water.</span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>9-18-13</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>gibson reaction
+with KR2 G-blocks</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'><span
+style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>-5ul
+concentrated plasmid backbone</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'><span
+style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>-1ul
+of each G-block</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'><span
+style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>-3ul
+H2O</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'><span
+style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>-10ul
+x Gibson Master Mix</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'><span
+style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>-PCR
+Machine 50C for 1 hr.</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>chemicompetent
+transformation of e.coli with Gibson reaction products.</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>plate
+transformed e.coli on LB+Chloramphenicol plates. (400ul , 200ul, 100ul, and one
+with untransformed ecoli)</span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>9-19-13</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>suspend
+colonies in mili q water</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>mili q water
+with colonies colony PCR on colonies from 400ul and 200ul plates</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>15 cycles</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>94 C initial
+denature 5 minutes</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>94 C denature
+s</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>55 C first
+anneal 30s</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>68 C extension
+:00</span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>15 cycles
+anneal drop down 7 C</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>94 C denature
+:15</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>48 C second
+anneal<span style="mso-spacerun: yes">&nbsp; </span>:30</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>68 C extension
+:00</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>68 C final
+extension 5:00</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>4 C hold
+infinite</span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>inoculation of
+e. coli colonies in LB + chloramphenicol </span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>9-20-13</span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>run gel on PCR
+products from 9-19-13</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>gel legend: 1Kb
+ladder, “400”, “200”, negative control (no template)</span></p>
+<p class=normal><span style='mso-no-proof:yes'><!--[if gte vml 1]><v:shape
+ id="image07.jpg" o:spid="_x0000_i1025" type="#_x0000_t75" style='width:232pt;
+ height:279pt;visibility:visible;mso-wrap-style:square'>
+ <v:imagedata src="pumpsprotocolscaulobacterexpressionprotocols_files/image019.jpg"
+  o:title=""/>
+ <v:textbox style='mso-rotate-with-shape:t'/>
+</v:shape><![endif]--><![if !vml]><img width=234 height=281
+src="pumpsprotocolscaulobacterexpressionprotocols_files/image020.gif" v:shapes="image07.jpg"><![endif]></span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>Zymo research
+plasmid midi-prep from e. coli cultures</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>9-21-13</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>Send psb1c3+KR2
+plasmid in for sequencing.</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>9-24-13</span></p>
+<p class=normal><span style='font-size:12.0pt;line-height:115%'>receive
+sequencing results (not sure how to explain the results)</span></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+<p class=normal><o:p>&nbsp;</o:p></p>
+</div>
+</body>
+</html>