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Team:Exeter/lab book/gibs/wk5
From 2012.igem.org
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<!<p><b>**Monday 6.8.12**</b></p><br> | <!<p><b>**Monday 6.8.12**</b></p><br> | ||
| - | <p><a href="https://2012.igem.org/Team:Exeter/lab_book/proto/4" style="color:#1d1d1b"><u>3A BioBrick assembly</u></a> of RBS- | + | <p><a href="https://2012.igem.org/Team:Exeter/lab_book/proto/4" style="color:#1d1d1b"><u>3A BioBrick assembly</u></a> of RBS-<i>wbbC</i>(d) (BBa_B0034 + BBa_K764005)</p><br><p> |
Upstream</p><p> | Upstream</p><p> | ||
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Water - 14.4 µl</p><br><p> | Water - 14.4 µl</p><br><p> | ||
| - | Downstream | + | Downstream <i>wbbC</i>(d) </p><p> |
DNA - 2.8 µl</p><p> | DNA - 2.8 µl</p><p> | ||
Water - 19.7 µl</p><br><p> | Water - 19.7 µl</p><br><p> | ||
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**Wednesday 8.8.12**</p><br> | **Wednesday 8.8.12**</p><br> | ||
| - | <p><a href="https://2012.igem.org/Team:Exeter/lab_book/proto/1" style="color:#1d1d1b"><u>Transformed </u></a> RBS- | + | <p><a href="https://2012.igem.org/Team:Exeter/lab_book/proto/1" style="color:#1d1d1b"><u>Transformed </u></a> RBS-<i>wbbC</i>(d) (BBa_B0034 + BBa_K764005)</p> |
<p>2 µl into 25 µl competent cells</p><p> | <p>2 µl into 25 µl competent cells</p><p> | ||
50 µl and 150 µl LB(chlor) spread plates made</p><br><p> | 50 µl and 150 µl LB(chlor) spread plates made</p><br><p> | ||
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<b>**Thursday 9.8.12**</b></p><br> | <b>**Thursday 9.8.12**</b></p><br> | ||
| - | <p><a href="https://2012.igem.org/Team:Exeter/lab_book/proto/2" style="color:#1d1d1b"><u>Transferred </u></a> RBS- | + | <p><a href="https://2012.igem.org/Team:Exeter/lab_book/proto/2" style="color:#1d1d1b"><u>Transferred </u></a> RBS-<i>wbbC</i>(d) (BBa_B0034 + BBa_K764005) transformed colonies to LB(chlor) medium</p><br><p> |
<b>**Friday 10.8.12**</b></p><br><p> | <b>**Friday 10.8.12**</b></p><br><p> | ||
| - | <p> RBS- | + | <p> RBS-<i>wbbC</i>(d) transformed cells broth tubes centrifuged for 5 minutes, 3901 rcf, 4 °C</p> |
<p>Supernatant discarded</p> | <p>Supernatant discarded</p> | ||
<p>Miniprep according to<a href="http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_k0502.pdf" style="color:#57B947" target="_blank"><u>Fermentas protocol</u></a></p> | <p>Miniprep according to<a href="http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_k0502.pdf" style="color:#57B947" target="_blank"><u>Fermentas protocol</u></a></p> | ||
Revision as of 17:45, 25 September 2012
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Operon Construction: 6th - 10th August 2012 **Monday 6.8.12**3A BioBrick assembly of RBS-wbbC(d) (BBa_B0034 + BBa_K764005) Upstream RBS 3 (BBa_B0034) plasmid DNA - 8.1 µl Water - 14.4 µl Downstream wbbC(d) DNA - 2.8 µl Water - 19.7 µl
Destination plasmid pSB1C3 (chloramphenicol resistant) Upstream: 2 µl Downstream: 2 µl MilliQ water 2.5 23 °C (room temp.) incubation for 1 hour 20 minute heat kill 80 °C **Wednesday 8.8.12** Transformed RBS-wbbC(d) (BBa_B0034 + BBa_K764005) 2 µl into 25 µl competent cells 50 µl and 150 µl LB(chlor) spread plates made **Thursday 9.8.12** Transferred RBS-wbbC(d) (BBa_B0034 + BBa_K764005) transformed colonies to LB(chlor) medium **Friday 10.8.12**
RBS-wbbC(d) transformed cells broth tubes centrifuged for 5 minutes, 3901 rcf, 4 °C Supernatant discarded Miniprep according toFermentas protocol Step 1045µl milli-Q water added in place of elution buffer, followed by another 5 µl after incubation and centrifugation |
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