IGEM:Groningen/Notebook/iGEM 2011/2011/07/08: Difference between revisions

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==Entry title==
==*8th of July==
* Insert content here...
<br>Colony PCR of  transformants to check if any transformant contains the plasmid with cI-LVA and LasR-LVA.
<br>20μl total volume was used and the PCR was done with taq polymerase.
<br>PCR program:
<br>Denaturation: 95 °C for ten minutes
<br>Cycle (33×):
<br> Denaturation:  95°C for 30 seconds
<br> Annealing:    60°C for 30 seconds
<br> Extension:    72°C for 2 minutes
<br>Final extension: 72°C for ten minutes
<br>Store at 4 °C infinite
<br>
<br>PCR with the new pBAD primers (dissolved and dilution made by me)
<br>PCR was done in total colume of 50μl and with tag and Pfu polymerase (ratio 6:1) so Pfu can correct the mistakes that taq made
<br>Denaturation: 94°C for ten minutes
<br>Cycle (30×)
<br> Denaturation: 94°C for 30 seconds
<br> Annealing: 62°C and 65°C for 30 seconds
<br> Extension: 72°C for 2 minutes
<br>Final extension: 72°C for ten minutes
<br>Store at 4 °C infinite
<br>
<br>Check them on agarose gel 1% (TBE).
<br>After that: Clean up the pBAD samples and nanodrop the samples
<br>Nanodrop results:
<br>




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*8th of July


Colony PCR of transformants to check if any transformant contains the plasmid with cI-LVA and LasR-LVA.
20μl total volume was used and the PCR was done with taq polymerase.
PCR program:
Denaturation: 95 °C for ten minutes
Cycle (33×):
Denaturation: 95°C for 30 seconds
Annealing: 60°C for 30 seconds
Extension: 72°C for 2 minutes
Final extension: 72°C for ten minutes
Store at 4 °C infinite

PCR with the new pBAD primers (dissolved and dilution made by me)
PCR was done in total colume of 50μl and with tag and Pfu polymerase (ratio 6:1) so Pfu can correct the mistakes that taq made
Denaturation: 94°C for ten minutes
Cycle (30×)
Denaturation: 94°C for 30 seconds
Annealing: 62°C and 65°C for 30 seconds
Extension: 72°C for 2 minutes
Final extension: 72°C for ten minutes
Store at 4 °C infinite

Check them on agarose gel 1% (TBE).
After that: Clean up the pBAD samples and nanodrop the samples
Nanodrop results:


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