Team:TUDelft/12 August 2009

From 2009.igem.org

(Difference between revisions)
(→Calin)
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No growth for assembly CB. CE has many colonies. pKD plasmids all have growth, plates put into fridge.
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.
{{Template:TUDelftiGEM2009_end}}
{{Template:TUDelftiGEM2009_end}}

Revision as of 16:36, 12 August 2009

Lab Notebook

July
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August
MTWTFSS
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September
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October
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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 --- --- ---

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.

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