Lab Notebook

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 --- --- ---

trbK_KO_PCR --- --- ---

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.