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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 (part 3) 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 was due to the incorrect Tm. We also performed a sixth PCR tube 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 that the chromosomal DNA extraction from ''Bacillus subtilis'' ATCC 6633 had worked. | + | We found that the PCR tube containing the ''spaIFEG'' gene (part 3) 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 was due to the incorrect Tm. We also performed a sixth PCR tube as a control, which contained the ''ara'' forward and reverse primers (we previously 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 that the chromosomal DNA extraction from ''Bacillus subtilis'' ATCC 6633 had worked. |
| - | We have cut the Plasmid Vector (part 1), Promoter & RBS (part 2) and Double terminator (part 4) gel bands | + | We have cut the Plasmid Vector (part 1), Promoter & RBS (part 2) and Double terminator (part 4) gel bands following the [[Team:Newcastle/Gel_extraction| gel extraction]] protocol. We will wait until the ''spaIFEG'' gene (part 3) has been cut from the gel. This will be carried out on [[Team:Newcastle/12 August 2010#Subtillin_immunity_BioBrick| tommorrow]]. After all four parts have been cut from gels, the[[Team:Newcastle/Gel_extraction| gel extraction]] protocol will be completed. Once all four parts have been successfully extracted, a single PCR tube will be prepared for the final step for the Gibson cloning. |
==Conclusion== | ==Conclusion== | ||
Revision as of 08:40, 13 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 (part 3) 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 was due to the incorrect Tm. We also performed a sixth PCR tube as a control, which contained the ara forward and reverse primers (we previously 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 that the chromosomal DNA extraction from Bacillus subtilis ATCC 6633 had worked.
We have cut the Plasmid Vector (part 1), Promoter & RBS (part 2) and Double terminator (part 4) gel bands following the gel extraction protocol. We will wait until the spaIFEG gene (part 3) has been cut from the gel. This will be carried out on tommorrow. After all four parts have been cut from gels, the gel extraction protocol will be completed. Once all four parts have been successfully extracted, a single PCR tube will be prepared for the final step for the Gibson cloning.
Conclusion
Go back to our main Lab book page
   
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