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Team:TUDelft/12 August 2009
From 2009.igem.org
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Checked concentrations: | Checked concentrations: | ||
| - | oriT-R_KO_PCR | + | oriT-R_KO_PCR 72.8 1.93 1.78 |
| - | trbK_KO_PCR | + | trbK_KO_PCR 34.5 1.70 1.17 |
Sriram transformed CB and CC with heat shock on CAM plates. | Sriram transformed CB and CC with heat shock on CAM plates. | ||
Revision as of 11:07, 13 August 2009
Lab Notebook
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12 August 2009
Calin
No growth for assembly CB. CE has many colonies. pKD plasmids all have growth, plates put into fridge.
Did a digest on trbK with X+P (4 uL DNA). Disest on GFP-gen with E+S (5 uL DNA). Digest on pTet-RBS E+S (8 uL DNA). Did ligations CB (GFPgen+oriTR on pSB1C3) and CC (pTet-RBS+trbK on pSB1C3).
Rehydrated knockout primers to make 100uM stock. Made the linear fragments needed for the knockout. Made 10uM primer mix for both oriT_KO_PCR and trbK_KO_PCR.
Made master mix for Pfx platinum PCR.
Ran both oriT_KO_PCR and trbK_KO_PCR PCR reactions using pKD4 plasmid as template.
Did a PCR purify. On the trbK_KO_PCR sample the pH was too high. Added sodium acetate and a tiny bit of acetic acid to lower pH. Did a PCR purify using the Qiaquick kit.
Checked concentrations:
oriT-R_KO_PCR 80 1.88 1.93
trbK_KO_PCR 41.6 1.79 1.14
Ran a DpnI digestion on both. Enzyme used is old. Still working?
Ran another PCR purify.
Checked concentrations:
oriT-R_KO_PCR 72.8 1.93 1.78
trbK_KO_PCR 34.5 1.70 1.17
Sriram transformed CB and CC with heat shock on CAM plates.
Made another TRI+AMP plate with pKD46 and R751 for the knockout. Also made a 5mL tube culture.
