Team:TUDelft/12 August 2009

=12 August 2009=

Sriram
Not all the assembles worked in Chemical transformation and electro transformation since the gel gave different bands (check yesterday). We thought whether we would be able to combine cells got from both transformation. But after discussion with my supervisors we decided to redo the assemblies which were conflicting in the transformed cells. I planned to continue the restriction and ligation of the the assemblies 1, 3, 5, 6, 7 and 8. (Check this page for details of these assemblies' numbers)

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.

Daniel
Today I did miniprep on the tube cultures which all had growth. Besides I did glycerol stocks for future generations of TUDelft's iGEM teams. After miniprep, I checked the DNA's concentration (which somehow I always got low concentrations compared with the other members, you will see).

DNA:

Furthermore I did the digestion with biobrick assembly kit for all the biobricks I need for the "new" strategy and loc/key characterization. These digestions were analyzed with agarose 2% gel electrophoresis. All the sizes are correct (wait for the el image).