IGEM:Cambridge/2008/Notebook/Bacillus/2008/08/28

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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 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!