Imperial College/3 September 2008
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*GFP-Terminator biobrick and RFP-Terminator biobrick cut again with ''EcoRI'' and ''SpeI'' to produce biobrick vectors for our GeneArt produced clones | *GFP-Terminator biobrick and RFP-Terminator biobrick cut again with ''EcoRI'' and ''SpeI'' to produce biobrick vectors for our GeneArt produced clones | ||
*Biobrick digest run on a gel for gel purification of vector | *Biobrick digest run on a gel for gel purification of vector | ||
+ | *GeneArt constructs in vectors midiprepped from Xl1-Blue cells | ||
===Transformation of ''B.subtilis''=== | ===Transformation of ''B.subtilis''=== | ||
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*Lane 5- Taq PCR reaction, first 10 cycles at 56<sup>o</sup>C and then 20 cycles at 65<sup>o</sup>C using LacI gene primers but no template DNA (negative control) | *Lane 5- Taq PCR reaction, first 10 cycles at 56<sup>o</sup>C and then 20 cycles at 65<sup>o</sup>C using LacI gene primers but no template DNA (negative control) | ||
<br> | <br> | ||
- | *Lane 6- Taq PCR reaction, first 10 cycles at | + | *Lane 6- Taq PCR reaction, first 10 cycles at 54<sup>o</sup>C and then 20 cycles at 60<sup>o</sup>C using Aad9 primers, |
- | *Lane 7- Taq PCR reaction, first 10 cycles at 52<sup>o</sup>C and then 20 cycles at | + | *Lane 7- Taq PCR reaction, first 10 cycles at 52<sup>o</sup>C and then 20 cycles at 60<sup>o</sup>C using Aad9 primers, |
- | *Lane 8- Taq PCR reaction, first 10 cycles at | + | *Lane 8- Taq PCR reaction, first 10 cycles at 50<sup>o</sup>C and then 20 cycles at 60<sup>o</sup>C using Aad9 primers, |
- | *Lane 9 - Taq PCR reaction, first 10 cycles at | + | *Lane 9 - Taq PCR reaction, first 10 cycles at 50<sup>o</sup>C and then 20 cycles at 60<sup>o</sup>C using Aad9 primers but no template DNA |
*As can be observed all the PCRs failed, including the positive control. The most likely explanation is that the reactions were left to long before PCR started (in the case of Pfu) or that a vital reaction component was not correctly added to the reactions. A trial of Pfu will need to be carried out to determine if the enzyme itself may be a problem | *As can be observed all the PCRs failed, including the positive control. The most likely explanation is that the reactions were left to long before PCR started (in the case of Pfu) or that a vital reaction component was not correctly added to the reactions. A trial of Pfu will need to be carried out to determine if the enzyme itself may be a problem | ||
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===Motility=== | ===Motility=== | ||
*Manual tracking of synthetic data was continued. Error bars were plotted and uploaded onto the OWW Wiki. | *Manual tracking of synthetic data was continued. Error bars were plotted and uploaded onto the OWW Wiki. | ||
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+ | <br> | ||
+ | {{Imperial/EndPage|Notebook|Notebook}} |
Latest revision as of 20:44, 28 October 2008
3 September 2008WetlabCloning
Transformation of B.subtilis
ResultsUpper Lanes:
Lower Lanes:
Dry LabMotility
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