Team:Exeter/lab book/gibs/wk5

From 2012.igem.org

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     <!------------ENTER LAB BOOK HERE: PLEASE INCLUDE DATES WITHIN THE WEEK------------>  
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     <!<p>**Monday 6.8.12**</p><br>
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<p><a href="https://2012.igem.org/Team:Exeter/lab_book/proto/4" style="color:#1d1d1b"><u>3A BioBrick assembly</u></a> of RBS-WbbC(d) (BBa_B0034 + BBa_K764005)</p><br><p>
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Upstream</p><p>
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RBS 3 (BBa_B0034) plasmid DNA - 8.1 µl</p><p>
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Water - 14.4 µl</p><br><p>
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Downstream WbbC(d) </p><p>
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DNA  - 2.8 µl</p><p>
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Water  - 19.7 µl</p><br><p>
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Destination plasmid pSB1C3 (chloramphenicol resistant) <ahref="https://2012.igem.org/Team:Exeter/lab_book/proto/10" style="color:#1d1d1b"><u>Digestion</u></a></p><p>
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<a href="https://2012.igem.org/Team:Exeter/lab_book/proto/10" style="color:#1d1d1b"><u>Ligation</u></a></p><br><p>
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Upstream: 2 µl</p><p>
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Downstream: 2 µl</p><p>
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MilliQ water 2.5</p><br><p>
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23 °C (room temp.) incubation for 1 hour</p><p>
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20 minute heat kill 80 °C</p><br><p>
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**Wednesday 8.8.12**</p><br>
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<p><a href="https://2012.igem.org/Team:Exeter/lab_book/proto/1" style="color:#1d1d1b"><u>Transformed </u></a> RBS-WbbC(d) (BBa_B0034 + BBa_K764005)</p>
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<p>2 µl into 25 µl competent cells</p><p>
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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>
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<p><a href="https://2012.igem.org/Team:Exeter/lab_book/proto/2" style="color:#1d1d1b"><u>Transferred </u></a> RBS-WbbC(d) (BBa_B0034 + BBa_K764005) transformed colonies to LB(chlor) medium</p><br><p>
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<b>**Friday 10.8.12**</b></p><br><p>
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<p> RBS-WbbC(d) transformed cells broth tubes centrifuged for 5 minutes, 3901 rcf, 4 °C</p>
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<p>Supernatant discarded</p>
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<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>
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<p><b><i>Step 10</i></b>45µl milli-Q water added in place of elution buffer, followed by another 5 µl after incubation and centrifugation</p>
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Revision as of 17:22, 25 September 2012

ExiGEM2012 Lab Book Gibs wk5

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) Digestion

Ligation


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

Retrieved from "http://2012.igem.org/Team:Exeter/lab_book/gibs/wk5"