IGEM:Cambridge/2008/Notebook/Turing Pattern Formation/2008/08/28

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(Results from Bacillus transformation with glycerol stocks)
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Revision as of 07:50, 5 September 2008



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Results from transformation of ligation products

- nothing on plate, even on control plate

  • Reasons ?

- cells non competent anymore (try with fresh competent cells)

- not enough DNA (try with 10μL of DNA)

- very short ligation products

Results from Bacillus transformation with glycerol stocks

Strain added vector Antibiotic number of colonies Conclusion
IA771 none blank plate confluent colonies cells are alive
IA771 none Cm5 0 no contamination
IA771 ECE166 Cm5 2 transformation ok, very low efficiency
IA751 none blank plate confluent colonies cells are alive
IA751 none Cm5 0 no contamination
IA751 none Spc50 0 no contamination
IA751 ECE112 Cm5 0 problem, we can not manage to transform this vector
IA751 ECE153 Spc50 156 transformation ok, good efficiency

Transformation of ligation products (new)

  • Products to ligate : Pupp, Pspac, agrD, RBS S and RBS W

- new fresh competent TOP 10

- 5μL of DNA (1.5μL of PUC9)

- 2h30 in the incubator

Test AmyE insertion

  • Colonies PCR for Bacillus (IA751 transformed with ECE153, IA751, and IA771)

- 20μL of lysozyme, a few colonies for each

- Cycle : 15min qt 37°C, 15min at 99°C, 1min at 4°C, 1min at 99°C and 1min at 4°C

- for each PCR : 12μL of cells, 1μL of cells, 1μL of primer1, 1μL of primer2, 5μL of master mix

- PCR (iGEM program)

- run a gel

  • Result : nothing on the gel!!! But we can not manage to PCR anything... there is a problem in our PCR protocol, or products!


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