Team:Newcastle/12 August 2010

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Contents

rocF Gibson Cloning

Discussion

Today we have colonies from our transformation yesterday. Overnight cultures will be done for DNA extraction tomorrow.

Subtilin Immunity BioBrick

Aims

The aims for today are to perform gel electrophoresis again, and then gel extraction.

Materials and protocol

Please refer to the gel electrophoresis and the gel extraction protocols.

Results

Discussion

We found that the PCR tube containing the spaIFEG gene did not have any band, even though another gel electrophoresis was performed (at 63°C) again. We decided to perform five different PCR reactions at different temperatures: 46°C, 51°C, 56°C, 61°C and 66°C - this is to check whether the missing band occured was due to the incorrect Tm. We also performed a sixth PCR tube used as a control, which contained the ara forward and reverse primers (we had extracted from Bacillus subtilis ATCC 6633 on 7th July 2010) - the Tm for this was 59°C, with the extention time of fifteen seconds (specific for ara primers). ara primers were put into the tube because they were the primers that confirmed the chromosomal DNA extraction from Bacillus subtilis ATCC 6633.

We have cut the Plasmid Vector, Promoter & RBS and Double terminator gel bands, following the gel extraction protocol.

Conclusion

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