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Team:Exeter/lab book/gibs/wk2
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| - | <a href="https://2012.igem.org/Team:Exeter/ | + | <a href="https://2012.igem.org/Team:Exeter/Results#usepoly"; style="color:#e30614" target="_blank"><font size="3"><b>Results: Part 1</b></font></a> |
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</p> | </p> | ||
| - | <a href="https://2012.igem.org/Team:Exeter/ | + | <a href="https://2012.igem.org/Team:Exeter/Results/GvsB"; style="color:#e30614" target="_blank"><font size="3"><b>Results: Part 2</b></font></a> |
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<!--End Project Division Week Hyperlinks--> | <!--End Project Division Week Hyperlinks--> | ||
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<!<p><b>**Monday 16.7.12**</b></p><br> | <!<p><b>**Monday 16.7.12**</b></p><br> | ||
| - | <p>TetrRBS and RBS (BBa_B0034) <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/6" style="color:# | + | <p>TetrRBS and RBS (BBa_B0034) <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/6" style="color:#57b947"><u>BioBricks resuspended</u></a> and used for <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/1" style="color:#1d1d1b"><u>competent cell transformation.</u></a></p> |
<p>1µl of resuspended DNA added to 25µl top10 competent cells.</p> | <p>1µl of resuspended DNA added to 25µl top10 competent cells.</p> | ||
<p>LB(amp) (1:1000) plates used for 20µl and 100µl spread plates.</p><br> | <p>LB(amp) (1:1000) plates used for 20µl and 100µl spread plates.</p><br> | ||
<p><b>**Tuesday 17.7.12**</b></p><br> | <p><b>**Tuesday 17.7.12**</b></p><br> | ||
| - | <p>Previous days spread plate colonies <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/2" style="color:# | + | <p>Previous days spread plate colonies <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/2" style="color:#57b947"><u>transferred to liquid medium,</u></a> 1:1000 LB(amp) broth.</p><br> |
<p><b>**Wednesday 18.7.12**</b></p><br> | <p><b>**Wednesday 18.7.12**</b></p><br> | ||
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<p>Miniprep attempt from <a href="http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_k0502.pdf" style="color:#57B947" target="_blank"><u>protocol</u></a></p> | <p>Miniprep attempt from <a href="http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_k0502.pdf" style="color:#57B947" target="_blank"><u>protocol</u></a></p> | ||
<p>Pellet was considered too small, believed to be due to shaking incubator turning off overnight</p> | <p>Pellet was considered too small, believed to be due to shaking incubator turning off overnight</p> | ||
| - | <p>4 colonies were <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/2" style="color:# | + | <p>4 colonies were <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/2" style="color:#57b947"><u>transferred to liquid medium</u></a> as on previous day</p><br> |
<p><b>**Thursday 19.7.12**</b></p><br> | <p><b>**Thursday 19.7.12**</b></p><br> | ||
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<p>Eluted DNA put on ice for analysis with nanodrop spectrophotometer and then stored at -20 °C</p><br> | <p>Eluted DNA put on ice for analysis with nanodrop spectrophotometer and then stored at -20 °C</p><br> | ||
| - | <p>Plasmids from miniprep digested with EcoR1-HF and PstI according to <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/4" style="color:# | + | <p>Plasmids from miniprep digested with EcoR1-HF and PstI according to <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/4" style="color:#57b947"><u>digestion protocol</u></a></p><br> |
<p><ul>Plasmid DNA - 6.5 µl (500ng from largest volume)</p> | <p><ul>Plasmid DNA - 6.5 µl (500ng from largest volume)</p> | ||
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Latest revision as of 00:09, 27 September 2012
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Operon Construction: 16th - 20th July 2012 **Monday 16.7.12**TetrRBS and RBS (BBa_B0034) BioBricks resuspended and used for competent cell transformation. 1µl of resuspended DNA added to 25µl top10 competent cells. LB(amp) (1:1000) plates used for 20µl and 100µl spread plates. **Tuesday 17.7.12** Previous days spread plate colonies transferred to liquid medium, 1:1000 LB(amp) broth. **Wednesday 18.7.12** Centrifuged tubes, 4℃, 10 minutes, 3901 rcf Miniprep attempt from protocol Pellet was considered too small, believed to be due to shaking incubator turning off overnight 4 colonies were transferred to liquid medium as on previous day **Thursday 19.7.12** Transferred cultures to identical falcon tubes to remove pipette tips Centrifuged at 3901 rcf for 5 minutes, 4 °C Supernatant tipped away Miniprep attempt 2 Step 10 50 µl elution buffer added Incubated for 2 minutes at room temperature Centrifuged for 2 minutes, 13000 rpm 20 µl milli-Q water added Centrifuged for 2 minutes, 13000 rpm Columns discarded Eluted DNA put on ice for analysis with nanodrop spectrophotometer and then stored at -20 °C Plasmids from miniprep digested with EcoR1-HF and PstI according to digestion protocol
EcoR1-HF - 1 µl PstI - 1 µl 10x NEBuffer 2 - 5 µl 100x BSA - 0.5 µl MQ Water - 36 µl Incubated for 10 minutes at 37 °C DNA run on gel to test fragments |
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