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Team:Newcastle/12 August 2010
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==Discussion== | ==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 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 [[Team:Newcastle/7_July_2010#Chromosomal_prep| 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 [[Team:Newcastle/Gel_extraction| gel extraction]] protocol. | We have cut the Plasmid Vector, Promoter & RBS and Double terminator gel bands, following the [[Team:Newcastle/Gel_extraction| gel extraction]] protocol. | ||
Revision as of 14:33, 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
Go back to our main Lab book page
   
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