Team:Goettingen/NoteBook w6
From 2013.igem.org
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- | + | <div class="monat">July</div> | |
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<span class="date">12th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span> | <span class="date">12th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span> | ||
<div class="cont"> | <div class="cont"> | ||
- | <p class="timeline-title">Mini Plasmid Preparation, Test Digest of Clone 12 RBS-GFP-Terminator (see gel 11.07.) with EcoRI</p> | + | <p class="timeline-title goe-rt">Mini Plasmid Preparation, Test Digest of Clone 12 RBS-GFP-Terminator (see gel 11.07.) with EcoRI</p> |
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<span class="date">11th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span> | <span class="date">11th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span> | ||
<div class="cont"> | <div class="cont"> | ||
- | <p class="timeline-title">Plasmid mini-prep, Enyzme test: ThermoScientific vs. NEB (Biobrick Assembly Kit) ,Further RD of yesterday´s new protocol with part 7,</p> | + | <p class="timeline-title goe-rt">Plasmid mini-prep, Enyzme test: ThermoScientific vs. NEB (Biobrick Assembly Kit) ,Further RD of yesterday´s new protocol with part 7,</p> |
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+ | <p class="timeline-title goe-dac">Making 8 liters of expression culture of <i>L. monocytogenes</i> carrying gene for the DAC-domain for crystallization<br /></p> | ||
+ | <div class="timeline-cont"> | ||
+ | <p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family: | ||
+ | "Arial","sans-serif"">Making 8 liters of expression culture of <i>L. | ||
+ | monocytogenes </i>carrying gene for the DAC-domain for crystallization</span></b></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">50ml LB | ||
+ | medium containing Ampicillin inoculated with cryo 1013 stock</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">incubation | ||
+ | o/n at 37°C with agitation</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">measure | ||
+ | OD<sub>600</sub></span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">inoculation | ||
+ | of 1l LB medium containing Ampicillin to OD<sub>600</sub>=0,1</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">incubation | ||
+ | for 2 hours at 37°C to an OD<sub>600</sub>=0,5-0,9</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">induction | ||
+ | of expression by adding 1ml of 1M IPTG</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">incubation | ||
+ | for 12-14h at 16°C with agitation</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">measure | ||
+ | OD<sub>600</sub></span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">divide | ||
+ | 1l of culture in twice 500ml</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">centrifugation | ||
+ | at 5000 rpm for 10min at 4°C</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">resuspend | ||
+ | the pellets in ice cold 20ml buffer W and take it in a falcon tube</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">pool | ||
+ | both separated treated samples together</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">centrifugation | ||
+ | at 5000 rpm for 15min at 4°C</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">discard | ||
+ | supernatant </span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">store | ||
+ | pellet at -20°C</span></p> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif""> </span></p> | ||
+ | <p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family: | ||
+ | "Arial","sans-serif"">Protein purification</span></b></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">resuspend | ||
+ | cells from one falcon (pellet from 40ml of expression culture) in 10ml buffer | ||
+ | W</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">apply | ||
+ | solution from each falcon twice to french press</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">centrifugation | ||
+ | for 15min at 8500rpm at 4°C</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">transfer | ||
+ | supernatant into ultracentrifugation tubes</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">Ultracentrifugation | ||
+ | for 1h at 35000rpm at 4°C</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">Preparation | ||
+ | of columns: add 1ml of 50% Strep-Tactin and equilibrate with 10ml buffer W</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">Divide | ||
+ | supernatant received from ultracentrifugation into 2 equal volumes and fill | ||
+ | it up to 30ml with buffer W</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">Load | ||
+ | protein solution on prepared columns</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">Wash 5 | ||
+ | times with each 2,5ml buffer W</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">Elute | ||
+ | with 0,5ml (for eluate 1) or 1ml (eluate 2-4) of buffer E</span></p> | ||
+ | <p class="MsoNormal"><span lang="EN-US"> </span></p> | ||
+ | <p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family: | ||
+ | "Arial","sans-serif"">Determination of protein concentration via Bradford | ||
+ | test</span></b></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">Add 1ml | ||
+ | of Bradford solution (1:5 dilution = 200ml Bradford + 800ml H2O) to 3µl of | ||
+ | protein solution</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">Incubation | ||
+ | for 5min at RT</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">Measure | ||
+ | extinction at 595nm</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">Calculation: | ||
+ | c= OD595 / Vx0,0536</span></p> | ||
+ | <p class="MsoNormal" style="margin-left:36.0pt"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif""> </span></p> | ||
+ | <table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;border:none"> | ||
+ | <tbody><tr> | ||
+ | <td width="205" valign="top" style="width:153.5pt;border:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:11.0pt">Eluate</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border:solid windowtext 1.0pt; | ||
+ | border-left:none;padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:11.0pt">Absorption (nm)</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border:solid windowtext 1.0pt; | ||
+ | border-left:none;padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:11.0pt">C [µg/ml]</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="205" valign="top" style="width:153.5pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">1 E1</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">0,078</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">0,49</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="205" valign="top" style="width:153.5pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">2 E1</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">0,239</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">1,49</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="205" valign="top" style="width:153.5pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">3 E1</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">0,092</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">0,57</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="205" valign="top" style="width:153.5pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">4 E1</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">0,042</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">0,26</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="205" valign="top" style="width:153.5pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">5 E1</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">0,075</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">0,47</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="205" valign="top" style="width:153.5pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">6 E1</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">0,049</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">0,3</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="205" valign="top" style="width:153.5pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">7 E1</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">0,053</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">0,33</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="205" valign="top" style="width:153.5pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">8 E1</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">0,128</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">0,70</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="614" colspan="3" valign="top" style="width:460.6pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal" align="right" style="text-align:right"><span lang="EN-US" style="font-size:11.0pt">Total: 4,197 mg in 4ml</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="205" valign="top" style="width:153.5pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">1 E2</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">0,143</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">2,57</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="205" valign="top" style="width:153.5pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">2 E2</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">0,114</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">0,71</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="205" valign="top" style="width:153.5pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">3 E2</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">0,169</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">1,05</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="205" valign="top" style="width:153.5pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">4 E2</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">0,127</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">0,79</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="205" valign="top" style="width:153.5pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">5 E2</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">0,061</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">0,38</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="205" valign="top" style="width:153.5pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">6 E2</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">0,188</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">1,17</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="205" valign="top" style="width:153.5pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">7 E2</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">0,121</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">0,75</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="205" valign="top" style="width:153.5pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">8 E2</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">0,141</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">0,88</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="614" colspan="3" valign="top" style="width:460.6pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal" align="right" style="text-align:right"><span lang="EN-US" style="font-size:11.0pt">Total: 8,3 mg in 8ml</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody></table> | ||
+ | <p class="MsoNormal" style="margin-left:36.0pt"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif""> </span></p> | ||
+ | <p class="MsoNormal"><b><span lang="EN-US" style="font-size:12.0pt">Dialysis of | ||
+ | protein samples</span></b></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">Pool all | ||
+ | samples from E1 (4ml)</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">Pool all | ||
+ | samples from E2 (8ml)</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">Pool all | ||
+ | samples from E3 (8ml)</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">Transfer | ||
+ | eluates into dialysis bags</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">Put | ||
+ | dialysis bags into 4500ml H2O + 500ml 10x dialysis buffer</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif"">Dialysis | ||
+ | o/n with agitation</span></p> | ||
+ | <p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family: | ||
+ | "Arial","sans-serif""> </span></b></p> | ||
+ | |||
+ | |||
+ | <div class="fbutton">Fold ↑</div> | ||
</div> | </div> | ||
</div> | </div> | ||
- | + | </div> | |
+ | <br /> | ||
<div class="tlob" id="tl_0710"> | <div class="tlob" id="tl_0710"> | ||
<span class="date">10th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span> | <span class="date">10th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span> | ||
<div class="cont"> | <div class="cont"> | ||
- | <p class="timeline-title">Transformation from 8.7.13, colony PCR, Restriction Digest of Part 7 with new Protocol</p> | + | <p class="timeline-title goe-rt">Transformation from 8.7.13, colony PCR, Restriction Digest of Part 7 with new Protocol</p> |
<div class="timeline-cont"> | <div class="timeline-cont"> | ||
Line 2,883: | Line 3,297: | ||
</div> | </div> | ||
</div> | </div> | ||
- | + | <br /> | |
<div class="tlob" id="tl_0709"> | <div class="tlob" id="tl_0709"> | ||
<span class="date">09th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span> | <span class="date">09th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span> | ||
<div class="cont"> | <div class="cont"> | ||
- | <p class="timeline-title">DarRoperator PCR: 4 % gel run to check if PCR worked, preparation of antibiotics, test restriction.</p> | + | <p class="timeline-title goe-rt">DarRoperator PCR: 4 % gel run to check if PCR worked, preparation of antibiotics, test restriction.</p> |
<div class="timeline-cont"> | <div class="timeline-cont"> | ||
Line 3,529: | Line 3,943: | ||
product uncut; Lane 3: DarRop PCR product R.D. 1 µl; Lane 4: DarRop PCR product | product uncut; Lane 3: DarRop PCR product R.D. 1 µl; Lane 4: DarRop PCR product | ||
R.D. 5 µl; Lane 5: DarRop PCR product R.D. 10 µl</span></p> | R.D. 5 µl; Lane 5: DarRop PCR product R.D. 10 µl</span></p> | ||
- | + | ||
<p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt"><span lang="EN-US" style="font-family:Wingdings">è<span style="font:7.0pt "Times New Roman""> | <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt"><span lang="EN-US" style="font-family:Wingdings">è<span style="font:7.0pt "Times New Roman""> | ||
</span></span><span lang="EN-US">PCR product still detectable for 5 and 10 µl | </span></span><span lang="EN-US">PCR product still detectable for 5 and 10 µl | ||
Line 3,959: | Line 4,373: | ||
C3 R.D.; Lane 8: C4 uncut; Lane 9: C4 R.D.; Lane 10: C5 uncut; Lane 11: C5 R.D.; | C3 R.D.; Lane 8: C4 uncut; Lane 9: C4 R.D.; Lane 10: C5 uncut; Lane 11: C5 R.D.; | ||
Lane 12: 2 log ladder</span></p> | Lane 12: 2 log ladder</span></p> | ||
- | + | ||
<p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt"><span lang="EN-US" style="font-family:Wingdings">è<span style="font:7.0pt "Times New Roman""> | <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt"><span lang="EN-US" style="font-family:Wingdings">è<span style="font:7.0pt "Times New Roman""> | ||
</span></span><span lang="EN-US">Something strange of 100 bp got cloned…</span></p> | </span></span><span lang="EN-US">Something strange of 100 bp got cloned…</span></p> | ||
Line 3,969: | Line 4,383: | ||
</div> | </div> | ||
</div> | </div> | ||
- | + | <br /> | |
<div class="tlob" id="tl_0708"> | <div class="tlob" id="tl_0708"> | ||
<span class="date">08th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span> | <span class="date">08th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span> | ||
<div class="cont"> | <div class="cont"> | ||
- | <p class="timeline-title">Mini Plasmid Prep, PCR for DarRoperator, Ligation of Riboswitch inserts</p> | + | <p class="timeline-title goe-rt">Mini Plasmid Prep, PCR for DarRoperator, Ligation of Riboswitch inserts</p> |
<div class="timeline-cont"> | <div class="timeline-cont"> | ||
Line 5,553: | Line 5,967: | ||
</div> | </div> | ||
</div> | </div> | ||
+ | <br /> | ||
</html> | </html> |
Revision as of 20:02, 30 September 2013
![](https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png)
Mini Plasmid Preparation, Test Digest of Clone 12 RBS-GFP-Terminator (see gel 11.07.) with EcoRI
Mini Plasmid Preparation
-
3x 4 ml ON culture of plasmid part 7 clone 1
-
1x 4 ml ON culture of RBS-GFP-Terminator clone 12
-
With Macherey-Nagel kit
-
Elution in 30 µl HPLC water
-
Pooling of 3 samples of part 7
-
NanoDrop concentration measurement:
|
Concentration (ng/µl) |
A260nm/A280nm |
A260nm/A230nm |
Part 7 plasmid |
179.3 |
1.79 |
1.38 |
Plasmid RBS-GFP-Terminator C12 |
200.8 |
1.87 |
1.83 |
è Samples
stored at -20 °C (pink rag)
Test Digest of Clone 12 RBS-GFP-Terminator (see gel
11.07.) with EcoRI
Test Digest
pipette as follows:
1,5µl DNA (~300ng)
0.1µl EcoRI FD
1.5µl FD Buffer
11µl H20
àincubation at 37°C for 45 minutes
àAnalysis on 1% gel
Gel:
Marker|RBS-GFP-Terminator Clone12
The test RD shows
only one band indicating a successful cloning procedure. To be sure, the DNA
will be used for another transformation hopefully leading to clean clones.
-
the
plasmid DNA (RBS-GFP-TermC12) was analyzed on the 1%gel below.
2nd round of RD of DarR
and Ribiswitches A-D with new protocol
after Katrin Gunkas Überprotokoll
àFirst, the RDs of yesterday were
analyzed on a 1% gel for complete digestion.
Gel:
Marker|DarR|Riboswitch
40/41|40/43|42/43|42/41|-|RBS-GFP-TermC12|-|part7after2ndRound2ndElution
The Riboswitch
constructs shoe strong signals, DarR seems to be very sparsely left over.
àThe RDs were purified using the PCR purification kit
(500µl PB and elution in 25µl pre-warmed water)
àThe purified DNA was analyzed with nanodrop
nanodrop:
|
Concentration (ng/µl) |
A260nm/A280nm |
A260nm/A230nm |
DarR biobrick |
3.4 |
1.13 |
0.56 |
Riboswitch 40/41 |
9.1 |
1.63 |
1.08 |
Riboswitch 40/43 |
8.4 |
1.81 |
1.17 |
Riboswitch 42/41 |
5.7 |
2.26 |
1.00 |
Riboswitch 42/43 |
4.0 |
2.16 |
0.92 |
àOf the purified DNA, all 24µl left were used for the 2nd
round of RD with EcoRI FD
The 5 Reactions
were pipetted like this
24µl DNA
16µl Mastermix:
4µl FD buffer
4µl EcoRI FD
8µl H2O
àIncubation at 37°C for 45 minutes
àFrozen away at -20°C
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Plasmid mini-prep, Enyzme test: ThermoScientific vs. NEB (Biobrick Assembly Kit) ,Further RD of yesterday´s new protocol with part 7,
Miniprep of frozen plasmids from part7 and part8 in order to have DNA for
cloning
NanoDrop:
Sample |
ng/µl |
A260nm/A280nm |
A260nm/A230nm |
Part7
mini |
120.5 |
1.83 |
1.99 |
part8
mini |
113.9 |
1.86 |
2.11 |
Incubation
of Colony PCR master plates
è Plates
taken out and put ot 4 °C (luckily not overgrown)
Enyzme test:
ThermoScientific vs. NEB (Biobrick Assembly Kit)
è Since
PstI digest was incomplete, it might be that the enzymes don’t work properly
è Test
all enzymes in their preferred buffers
Test plasmid: plasmid part 1 purified on 1.7.13 à Linearization in single digest with
EcoRI(-HF), XbaI, SpeI or PstI
-
Reaction according to test restriction digest to
analyze clones after transformation
Component |
Volume |
Plasmid
part 1 (2 ng/µl) |
1 µl |
Enzyme |
1 µl |
10x
buffer |
1 µl |
dH2O |
7 µl |
Total
Volume |
10 µl |
è Preparation
of Mastermix:
Component |
Volume |
Plasmid part 1 |
15 µl |
dH2O |
105 µl |
è Preparation
of enzyme and buffer in Epi tubes
Company |
Enzyme |
Buffer |
Activity |
ThermoScientific |
EcoRI |
EcoRI buffer |
100 %;
recommended buffer |
EcoRI |
Buffer
0 |
100 % |
|
XbaI |
Tango |
100 % |
|
SpeI |
Tango |
100 % |
|
PstI |
Buffer
0 |
100 % |
|
NEB
(Biobrick Assembly Kit) |
EcoRI-HF |
CutSmart |
100 %;
recommended buffer |
EcoRI-HF |
NEB2.1 |
100 % |
|
XbaI |
CutSmart |
100 % |
|
XbaI |
NEB2.1 |
100 %;
recommended buffer |
|
SpeI |
CutSmart |
100 % |
|
SpeI |
NEB2.1 |
100 %;
recommended buffer |
|
PstI |
NEB3.1 |
100 %;
recommended buffer |
|
PstI |
NEB2.1 |
75 % |
è then
addition of 8 µl master mix
-
incubation at 37 °C for 2 h
-
gel run:
o
1 % agarose 1xTAE gel
o
3 µl 2 log ladder as a marker
o
Addition of 2 µl 5x LD to each reaction, then loading
of 8 µl
o
Run at 100 V
o
EtBr staining, destaining in water
o
UV detection
è All
digestions are partial; reaction of PstI
in NEB2.1 is less complete than other digests
è Does
not fit with complete digest of part 7 with EcoRI-HF in CutSmart buffer from
10.7.13; in this digest and in enzyme test different heating blocks were used à could
they be the problem?
Yesterday´s
Colony PCR of all constructs so far on 1% gel:
Gel:
Marker|Part8-plasmidDNA|GFP-Term-plasmid-DNA|Part8-w/o-insert-control|RBS-GFP-Term-C6|-7|-8|-9|-10|-11|-12|-13|-14|-15|-16|M
Marker|Part8-plasmidDNA|GFP-Term-plasmid-DNA|Part8-w/o-insert-control|RBS-GFP-Term-C17|-18|-19|part7-plasmidDNA|part7-w/o-insert
control-C1|-2|-3|DarR-biobrick-C1|-2|RiboswitchC-biobrick|RiboswitchD-biobrick|M
è Clone 12 from RBS-GFP-Terminator transformation could be positive,
at least partially
è Inoculation of clone 12 from RBS-GFP-Terminator transformation over
night + MiniPrep
è Re-transformation of plasmid, since clone seems to bee impure (band
for RBS-GFP-Terminator and RBS alone…)
Clones from RiboC and RiboD and DarR transformation are negative
Further
RD of yesterday´s new protocol with part 7
Yesterday´s samples were analyzed with the nanodrop:
NanoDrop
measurements from R.D. from yesterday:
Sample |
ng/µl |
A260nm/A280nm |
A260nm/A230nm |
purified
part7 R.D. PstI |
41.4 |
1.74 |
2.07 |
purified
part7 2nd elute PstI |
8.0 |
1.49 |
1.27 |
purified
part7 R.D. EcoRI |
4.2 |
1.35 |
0.77 |
purified
part7 2nd elute EcoRI |
2.0 |
0.84 |
0.51 |
Only the first elution of the RD with PstI
looks promising. This sample was used for the further, second digest. For time
reasons, the EcoRI digest was started in parallel to the 1% gel, that is
supposed to confirm the complete digestion of part 7 by PstI. The following
picture shows the progress of the digest at 30 additional minutes of RD (see
yesterday).
Gel:
Marker|PstI
RD Part 7
The digestion has not been completed, an
additional step has to be added in the end.
2nd
round of digest:
àpipetted one reaction (40µl) after
Katrin Gunkas Über-Protokoll (see yesterday) using EcoRI FD by Thermo
Scientific and the FD buffer:
20µl DNA (all that was left)
4µl FD Buffer
4µl EcoRI FD
12µl HPLC H2O
40µl reaction volume
àincubation at 37°C for 30 minutes
àsample put on ice and 3µl loaded on
1% gel.
àgel melted, samples lost
àconsidering the previous experience,
the digest was continued with EcoRI. Additionally, to proceed the unfinished
digest with PstI, that digest was added to the reaction.
The following things were added to the reaction:
2µl FD Buffer
3µl EcoRI FD
3µl PstI FD
12µl HPLC H2O
20µl leading to a 60 µl reaction (minus 3µl
on the gel)
àincubation at 37°C for 45 minutes
à3µl loaded on 1% gel (forgot to,
loaded 6µl with PB of purification kitàdidn’t
run)
Gel:
Needs
to be redone, I suggest with the second elution, so we don’t lose too much DNA.
àpurification of the sample using the
PCR purification kit, 500µl (different from protocol) PB buffer, 2x elution
with 25µl pre-warmed HPLC water
nanodrop
Sample |
ng/µl |
A260nm/A280nm |
A260nm/A230nm |
purified
part7 R.D. after 2nd round |
27 |
1.71 |
1.59 |
2nd
elution |
3.4 |
2.71 |
1.06 |
àstorage at -20°C, “white sticker Ute-Box”
Inoculation
of 4ml cultures for miniprep tomorrow
Three cultures of part 7 cryo-stocks from
11.6. and one culture of the RBS-GFP-Terminator construct Clone 12, which seems
to be carrying at least partly the right construct (see today´s gel). Incubated
in the Brutraum
New
1st round Single digestion of DarR and Ribiswitches A-D with new
protocol
after Katrin
Gunka Überprotokol
9 µl PCR product
4 µl 10X Buffer F.D.
4 µl PstI F.D.
23µl dH2O
incubated for 30 min at 37°C
Gel:
Wells as follows: DarR, Riboswitches 40/41,
40/43, 42/41, 42/43
rest of restriction stored in -20 freezer
(for further digestion more enzyme has to be added)
..But where is DarR?...
Making 8 liters of expression culture of L. monocytogenes carrying gene for the DAC-domain for crystallization
Making 8 liters of expression culture of L. monocytogenes carrying gene for the DAC-domain for crystallization
- 50ml LB medium containing Ampicillin inoculated with cryo 1013 stock
- incubation o/n at 37°C with agitation
- measure OD600
- inoculation of 1l LB medium containing Ampicillin to OD600=0,1
- incubation for 2 hours at 37°C to an OD600=0,5-0,9
- induction of expression by adding 1ml of 1M IPTG
- incubation for 12-14h at 16°C with agitation
- measure OD600
- divide 1l of culture in twice 500ml
- centrifugation at 5000 rpm for 10min at 4°C
- resuspend the pellets in ice cold 20ml buffer W and take it in a falcon tube
- pool both separated treated samples together
- centrifugation at 5000 rpm for 15min at 4°C
- discard supernatant
- store pellet at -20°C
Protein purification
- resuspend cells from one falcon (pellet from 40ml of expression culture) in 10ml buffer W
- apply solution from each falcon twice to french press
- centrifugation for 15min at 8500rpm at 4°C
- transfer supernatant into ultracentrifugation tubes
- Ultracentrifugation for 1h at 35000rpm at 4°C
- Preparation of columns: add 1ml of 50% Strep-Tactin and equilibrate with 10ml buffer W
- Divide supernatant received from ultracentrifugation into 2 equal volumes and fill it up to 30ml with buffer W
- Load protein solution on prepared columns
- Wash 5 times with each 2,5ml buffer W
- Elute with 0,5ml (for eluate 1) or 1ml (eluate 2-4) of buffer E
Determination of protein concentration via Bradford test
- Add 1ml of Bradford solution (1:5 dilution = 200ml Bradford + 800ml H2O) to 3µl of protein solution
- Incubation for 5min at RT
- Measure extinction at 595nm
- Calculation: c= OD595 / Vx0,0536
Eluate |
Absorption (nm) |
C [µg/ml] |
1 E1 |
0,078 |
0,49 |
2 E1 |
0,239 |
1,49 |
3 E1 |
0,092 |
0,57 |
4 E1 |
0,042 |
0,26 |
5 E1 |
0,075 |
0,47 |
6 E1 |
0,049 |
0,3 |
7 E1 |
0,053 |
0,33 |
8 E1 |
0,128 |
0,70 |
Total: 4,197 mg in 4ml |
||
1 E2 |
0,143 |
2,57 |
2 E2 |
0,114 |
0,71 |
3 E2 |
0,169 |
1,05 |
4 E2 |
0,127 |
0,79 |
5 E2 |
0,061 |
0,38 |
6 E2 |
0,188 |
1,17 |
7 E2 |
0,121 |
0,75 |
8 E2 |
0,141 |
0,88 |
Total: 8,3 mg in 8ml |
Dialysis of protein samples
- Pool all samples from E1 (4ml)
- Pool all samples from E2 (8ml)
- Pool all samples from E3 (8ml)
- Transfer eluates into dialysis bags
- Put dialysis bags into 4500ml H2O + 500ml 10x dialysis buffer
- Dialysis o/n with agitation
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Transformation from 8.7.13, colony PCR, Restriction Digest of Part 7 with new Protocol
Transformation from 8.7.13
- No clones on negative control
- No clones for Ribo A and B biobricks
- 3 clones for w/o insert control à partial digest of part 7; on the gel, a band of the uncut plasmid runs at approx. the same height as the band for pSB1C3 backbone à uncut plasmid was apparently extracted from the gel, as well…
- 2 clones for DarR biobrick
- 1 clone for Ribo C biobrick
- 1 clone for Ribo D biobrick
Partial digests, low number of positive clones, false positives…:
Biobrick Assembly Kit Cloning strategy = SHIT
è We’ll proceed according to old school, i.e. protocol “Katrin Gunka”…
Colony PCR
è To screen more efficiently for clones of RBS-GFP-Terminator cloning
è To screen transformation from 8.7.13 for positive clones
- For RBS-GFP-Terminator clones 6 – 19 and 1 clone from w/o insert ligation (part 8); additional controls: plasmid part 8; plasmid GFP-Terminator C1
- For single clone from cloning of Ribo D and Ribo C, both clones for DarR biobrick cloning and for all 3 clones from w/o insert ligation (part 7); additional control: plasmid part 7
- With primers VF2 and VR
- Prepare MasterMix (here: for 30 reactions)
Component |
1x reaction |
30x reaction |
Taq buffer 10x |
2.5 µl |
75 µl |
Taq Pol |
1 µl |
30 µl |
dNTP mix (12.5 mM each) |
1 µl |
30 µl |
Primer fwd (VF2) |
1 µl |
30 µl |
Primer rev (VR) |
1 µl |
30 µl |
dH2O |
18.5 µl |
555 µl |
Total |
25 µl |
750 µl |
è Distribute 25 µl of the master mix into the tubes
è For plasmid controls: preparation of 1:10 dilution of each plasmid in dH2O and addition of 1 µl of dilution to a tube; then addition of 25 µl master mix
- pick clones, streak out on a master plate with a toothpick, then use same toothpick to inoculate PCR reaction
- PCR protocol (30 cycles)
Step |
Temperature |
Time |
Initial denaturation |
94 °C |
5 min |
Denaturation |
94 °C |
45 sec |
Annealing |
54 °C |
40 sec |
Elongation |
72 °C |
4 min |
Final Elongation |
72 °C |
10 min |
Hold |
15 °C |
∞ |
- Incubation of master plate over day at 37 °C
Products stored at -20°C in a pipette-tip-stand
Restriction Digest of Part 7 with new Protocol
We now changed the protocol to single digest steps with purification instead of heat-inactivation.
Two digests were prepared, one with PstI and one with EcoRI-HF. For the double digest to be achieved, in a second digest, the products will be digested with the other enzyme, EcoRI-HF or PstI, respectively. This way, we try to find out, whether one enzyme inhibits the other enzyme´s function through its restriction. In the future, we will only need one of the two reactions, either starting PstI, or the other way around.
1st round of digest:
Two reactions were pipetted after Katrin Gunkas “Über-Protocol” as follows:
1: PstI digest
2: EcoRI-HF digest
1,5µg DNA (13µl for the isolated Part 7 DNA at 115.6ng/µl)
4µl Buffer (1: 3.1 for PstI / 2: CutSmart for EcoRI-HF)
4µl Enzyme (1: PstI by NEB / 2: EcoRI-HF by NEB)
19µl HPLC H2O
40µl reaction volume
àincubation at 37°C for 2h
à3µl on 1% gel to confirm complete digestion, undigested part 7 DNA as control (ATTENTION: Part 7 DNA almost empty)
Gel:
marker | uncut Part 7 | PstI RD | EcoRI RD
The EcoRI seems to be done cutting (although signal very low. Discussion: Since Part 7 was almost empty, I had to shake down the tube. This way the used DNA might have been diluted by condensed water, which I shook down). The PstI sample was put back onto the heat block at 37°C for 30 minutes with 3µl Enzyme added.
àpurification of the samples using the PCR purification kit, 500µl (different from protocol) PB buffer, 2x elution with 25µl pre-warmed HPLC water
àstorage at -20°C, “white sticker Ute-Box”
ànanodrop measurements have to be done tomorrow
è This R.D. was done using the NEB enzymes from the distribution kits. And all our cloning reactions using PstI did not work! In contrast, the test restriction digests that were done with the Fermentas enzymes EcoRI and PstI worked. Apparently, PstI enzyme from the distribution kit does not cut!
è Test PstI from distribution kit vs. Fermentas enzymes.
If this is true, then we have to prepare part 8 vector (SpeI, PstI cleavage) and GFP-Terminator cassette (XbaI, PstI cleavage) and all our inserts for RiboA, B, C, D and DarR biobrick again! Luckily, the PCR products and the plasmids are all at – 20 °C. So just another R.D. and another gel ex/ PCR clean-up…^^
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DarRoperator PCR: 4 % gel run to check if PCR worked, preparation of antibiotics, test restriction.
Preparation of plates from 500 ml LBCm plates (35 µg/ml Cm)
Further incubation of RBS-GFP-Terminator back-up plate
è Taken out in the evening (clones appear not green)
Cryostock of GFP-Term C1 overnight culture
stored at -80
Mini-prep of
Part7 C1
GFP-Term C1
RBS-GFP-Term C1
RBS-GFP-Term C2
RBS-GFP-Term C3
RBS-GFP-Term C4
RBS-GFP-Term C5
eluted in 30 µl HPLC
Nano Drop:
Sample |
ng/ µl |
260/280 |
260/230 |
GFP-Term gel ex |
6.8 |
1.84 |
0.10 |
pSB1C3 |
15.6 |
1.72 |
0.15 |
part 7 C1 |
115.3 |
1.91 |
2.22 |
GFP-Term C1 mini |
111.0 |
1.90 |
2.19 |
RBS-GFP-Term C1 |
285.7 |
1.89 |
2.26 |
RBS-GFP-Term C2 |
139.3 |
1.92 |
2.18 |
RBS-GFP-Term C3 |
157.6 |
1.88 |
2.04 |
RBS-GFP-Term C4 |
163.8 |
1.90 |
2.19 |
RBS-GFP-Term C5 |
155.0 |
1.91 |
2.20 |
DarRoperator PCR: 4 % gel run to check if PCR worked
Pouring of 4 % agarose-1xTAE-gel
1. Set waterbath to 70 °C and place gel chamber directly next to it.
2. Take 500 ml bottle (blue lid), add 4 g agarose and fill up to 100 ml with 1xTAE
3. Heat carefully in microwave (check every 5 – 10 sec if agarose does not … äh… überkochen… and mix by shaking bottle slightly (schwenken…) à Dissolving takes just a 2 – 4 min!
4. As soon as agarose is dissolved (clear solution, but many small bubbles from boiling and shaking making it turbid), place it in the waterbath (lid closed), wait for some time until bubbles are gone
5. Check again if agarose is still dissolved, if not, go back to step 3…
6. Pour gel rapidly.
Loading of
- 4 µl PCR reaction + 1 µl 5xLD
- 4 µl primer iGEM_36 + 1 µl 5xLD
- 4 µl primer iGEM_37 + 1 µl 5xLD
- 3 µl 2 log ladder (marker)
Run at 80 V
EtBr staining; destaining in water
UV detection
4% Gel with DarR operator:
Loading:
Lane 1: 2 log ladder; Lane 2: iGEM_36; Lane 3: iGEM_37; Lane 4: Negative control of PCR; Lane 4: PCR reaction tube 1; Lane 5: PCR reaction tube 2; Lane 6: PCR reaction tube 3; Lane 7: 2 log ladder
è PCR worked
è All reactions were pooled into one reaction
è Determination of concentration and purity (does actually not make sense…)
Sample |
Concentration (ng/µl) |
A260nm/A280nm |
A260nm/A230nm |
DarRop PCR product |
1063.5 |
1.65 |
1.11 |
è Far too much since PCR reaction, that was not purified: Calculate with 100 ng!
Restriction Digestion of DarRop PCR product for ligation with pSB1C3 obtained from R.D+gel ex of part 7
- More or less according to BioBrick Assembly Kit
Component |
Reaction 1 µl PCR product |
Reaction 5 µl PCR product |
Reaction 10 µl PCR product |
10x NEB2.1 buffer |
5 µl |
5 µl |
5 µl |
Eco-RI HF |
1 µl |
1 µl |
1 µl |
PstI |
1 µl |
1 µl |
1 µl |
PCR product |
1 µl |
5 µl |
10 µl |
dH2O |
42 µl |
38 µl |
33 µl |
Total |
50 µl |
50 µl |
50 µl |
è Reactions with 5 or 10 µl were incubated for 1 h at 37 °C
è Reaction with 1 µl was incubated for >1h at 37 °C
è Heat kill for all reactions: 20 min, 80 °C
- Gel run
- 4 % agarose-1xTAE gel
- Loading of
4 µl pf PCR product + 1 µl 5x LD as a control
8 µl of RD reactions + 2 µl 5x LD à just 8 µl of final sample (10 µl) loaded
3 µl 2 log ladder as a marker
- Run at 80 V
- EtBr staining, destaining in water
- UV detection
Gel:
Loading:
Lane 1: 2 log ladder; Lane 2: DarRop PCR product uncut; Lane 3: DarRop PCR product R.D. 1 µl; Lane 4: DarRop PCR product R.D. 5 µl; Lane 5: DarRop PCR product R.D. 10 µl
è PCR product still detectable for 5 and 10 µl reaction, but not for 1 µl reaction
è For 1 µl reaction probably band at bottom of lane à degradation?
è Use 5 and 10 µl reactions for cloning
è Reactions stored at -20 °C in pink box
Pooling of samples from gel ex on 4.7.13
- pSB1C3 vector from part 7
- GFP-Terminator insert
à determination of new concentration with NanoDrop
Test gel for plasmids and inserts
- plasmids for part 7 and GFP-Terminator C1 isolated in today’s MiniPrep
- plasmids part 1, 2, 3, 4 (purified on 2.7.13)
- GFP-Terminator insert after gel ex
- pSB1C3 vector obtained from RD and gel ex of part 7
- plasmid GFP-Terminator clone 1 (purified on 2.7.13)
- 1%-agarose-1xTAE gel
- Loading of around 30 ng (or more) of plasmid DNA (0.5 µl for all samples; exceptions: pSB1C3 vector from part 7 à 2 µl, GFP-Terminator insert à 4 µl, filled up with dH2O to 4 µl (exception: GFP-Terminator insert), then addition of 1 µl 5xLD
- Marker: 3 µl 2 log ladder
- Run at 100 V
- EtBr staining, destaining in water
- UV detection
Gel:
Loading:
Lane 1: 2 log ladder; Lane 2: part 1 uncut, Lane 3: part 2 uncut; Lane 4: part 3 uncut; Lane 5: part 4 uncut; Lane 6: part 7 (from today’s prep); Lane 7: gel ex pSB1C3 from part 7; Lane 8: GFP-Terminator C1 plasmid (former prep); Lane 9: GFP-Terminator C1 plasmid (today’s prep); Lane 10: gel ex GFP-Terminator construct from RD of GFP-Terminator C1 plasmid; Lane 11: 2 log ladder
è Part 7 is apparently impure à residual undigested plasmid though gel extraction was done (band of uncut plasmid has same height as pSB1C3 vector…)
Test Restriction Digestion of RBS-GFP-Terminator clones 1 – 5
- Fermentas/ThermoScientific enzymes were used
For 1 reaction:
Component |
Amount |
Plasmid DNA |
200 – 300 ng (1 – 1.5 µl in this case) |
PstI |
0.5 µl |
EcoRI |
0.5 µl |
dH2O |
Depends on plasmid amount (7 or 6.5 µl) |
10x buffer O |
1 µl |
Total |
10 µl |
è Preparation of plasmid amount in 3 µl total volume:
Plasmid from… |
…C1 |
…C2 |
…C3 |
…C4 |
…C5 |
Plasmid |
1 µl |
1.5 µl |
1.5 µl |
1.5 µl |
1.5 µl |
dH2O |
2 µl |
1.5 µl |
1.5 µl |
1.5 µl |
1.5 µl |
è Preparation of MasterMix
Component |
Amount |
PstI |
3 µl |
EcoRI |
3 µl |
dH2O |
30 µl |
10x buffer O |
6 µl |
Total |
42 µl |
è Add 7 µl to each reaction
- Incubation at 37 °C for 1.5 h (no heat kill necessary in this case, since it’s just run on the gel and then thrown away)
- Addition of 2.5 µl 5xLD
- Agarose gel electrophoresis
1%-agarose-1xTAE gel
Loading of 10 µl of C1 RD (well run over…), thus loading of 8 µl of other RD’s
Loading of mixture consisting of 0.5 µl of all uncut plasmids + 3.5 µl dH2O + 1 µl 5xLD as controls
Loading of 3 µl 2 log ladder as a marker
- Run at 100 V
- EtBr staining, destaining in water
- UV detection
Gel for Test restriction digestion of RBS-GFP-Terminator clones 1 - 5
Loading:
Lane 1: 2 log ladder; Lane 2: C1 uncut; Lane 3: C1 R.D.; Lane 4: C2 uncut; Lane 5: C2 R.D.; Lane 6: C3 uncut; Lane 7: C3 R.D.; Lane 8: C4 uncut; Lane 9: C4 R.D.; Lane 10: C5 uncut; Lane 11: C5 R.D.; Lane 12: 2 log ladder
è Something strange of 100 bp got cloned…
![](https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png)
Mini Plasmid Prep, PCR for DarRoperator, Ligation of Riboswitch inserts
Back-Up-Plates
è For
all inoculated RBS-GFP-Terminator clones
è Incubation
over day at 37 °C
MiniPlasmidPrep
è Clones
1 – 5 of RBS-GFP-Terminator construct
è Used
buffer A4 without Ethanol added… concentrations after elution were quite low…
è Elution
with 30 µl HPLC water (pre-warmed)
NanoDrop:
Sample |
Concentration (ng/µl) |
A260nm/A280nm |
A260nm/A230nm |
Clone 1 |
4.3 |
1.92 |
0.49 |
Clone 2 |
3.1 |
1.53 |
0.31 |
Clone 3 |
1.7 |
2.74 |
0.48 |
Clone 4 |
3.8 |
2.09 |
0.34 |
Clone 5 |
4.1 |
2.31 |
0.44 |
è Repeat
MiniPrep…
PCR for DarRoperator
è As
on 19.6.13
Component |
1x Reaction |
5x Master Mix |
5x
buffer HF |
10 µl |
50 µl |
PhuS |
1 µl |
- |
Primer
iGEM_36 |
4 µl |
20 µl |
Primer
iGEM_37 |
4 µl |
20 µl |
dNTPs |
2 µl |
145 µl |
dH2O |
29 µl |
10 µl |
Total |
50 µl |
245 µl à add 49 µl to each reaction |
Control: 1µl dH2O instead of PhuS polymerase
Protocol:
|
Temperature |
Time |
No. of cycles |
Initial Denaturation |
98.5
°C |
5min |
1 |
Denaturation |
98.5
°C |
30s |
10
cycles |
Annealing |
61.0°C |
30s |
|
Elongation |
72.0°C |
2min |
|
Final Elongation |
72.0°C |
10min |
1 |
Hold |
15
°C |
∞ |
1 |
è Reactions
stored at -20°C in 50 ml Falcon tube
Ligation of Riboswitch
inserts A, B, C, D with pSB1C3 backbone obtained from gel ex of dig. Part 7
plasmid
Ligation more or
less according to BioBrick-Assembly-Kit
Ca. 20 ng of pSB1C3 vector ligated with
ca. 20 ng of the different inserts
Reactions:
Component |
RiboA (iGEM_42/43) |
RiboB (iGEM_41/42) |
RiboC (iGEM_40/43) |
RiboD
(iGEM_40/41) |
DarR |
w/o
insert control |
MasterMix |
pSB1C3
vector (tube 3; 10.2 ng/µl) |
2 µl |
2 µl |
2 µl |
2 µl |
2 µl |
2 µl |
14 µl |
insert |
3.5 µl (5.9 ng/µl) |
1.5 µl (15.1 ng/µl) |
2 µl (11.7 ng/µl) |
3 µl (7.1 ng/µl) |
3 µl (6.8 ng/µl) |
- |
- |
dH2O/HPLC-H2O |
11.5 µl |
13.5 |
13 µl |
12 µl |
12 µl |
15 µl |
70 µl |
Ligase |
1 µl |
1 µl |
1 µl |
1 µl |
1 µl |
1 µl |
7 µl |
10x
ligation buffer |
2 µl |
2 µl |
2 µl |
2 µl |
2 µl |
2 µl |
14 µl |
Total |
20 µl |
20 µl |
20 µl |
20 µl |
20 µl |
20 µl |
105 µl |
è Prepare
5 µl of insert-HPLC water mix and add 15 µl of Master Mix
è Incubation
for 20 min at RT
è Heat
kill for 20 min at 80 °C
Transformation of E.coli DH5α competent cells
-
According to methods folder
Transformation of
part7-vector+Ribo
A
part7-vector+Ribo
B
part7-vector+Ribo
C
part7-vector+Ribo
D
part7-vector+DarR
part7-vector
w/o insert
Control
-
Whole ligation mix used
-
Negative control: 20 µl dH2O
-
Selection on LBCm
plates (35 µg/ml Cm)
Inoculation
of
RBS-GFP-Term C1, C2, C3, C4, C5
part7 C1
GFP-Terminator C1