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Team:Cambridge/Lab book/Week 6
From 2012.igem.org
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The remaining stray product had a slightly tricky secondary structure at the 3' end. It will be run at a series of annealing temperatures in a PCR machine capable of a temperature gradient. | The remaining stray product had a slightly tricky secondary structure at the 3' end. It will be run at a series of annealing temperatures in a PCR machine capable of a temperature gradient. | ||
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| + | '''[[Team:Cambridge/Protocols/Gibsonassembly|Construction of riboswitch plasmid with Gibson Assembly]]''' | ||
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| + | ---- | ||
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| + | *DNA from lanes 27+28, 27+29, 27+30 from gels run on Friday fused together with Gibson assembly to produce riboswitch construct without replacement of the first 8 codons of lac I with the 8 codons native to the gene downstream of the riboswitch. | ||
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| + | *DNA from lanes 22,23 and 24 fused with riboswitch DNA produced two weeks ago to produce riboswitch construct with replacement of the first 8 codons of lac I with the 8 codons native to the gene downstream of the riboswitch. | ||
'''[[Team:Cambridge/Protocols/TransformationofB.subtilis|Transformation of ''Bacillus'' with riboswitch construct]]''' | '''[[Team:Cambridge/Protocols/TransformationofB.subtilis|Transformation of ''Bacillus'' with riboswitch construct]]''' | ||
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| + | *Plasmids made by Gibson transformed into ''bacillus'' cells made two weeks ago and transformants plated out on 5μg/ml chloramphenicol plates. | ||
===Tuesday=== | ===Tuesday=== | ||
Revision as of 14:16, 31 July 2012
| Week: | 3 | 4 | 5 | 6 | 7 |
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Contents |
Monday
Not all products were obtained during Friday's PCR. Most of these missing products were large vector backbones. They are being run again, with a much longer extension time of 300s. If that fails, primers will be ordered to split the vectors into manageable chunks, and the PCR reattempted when they arrive.
PCR cycle x35:
- 15s Denaturing at 95 C
- 45s Annealing at 60 C
- 300s Extension at 72 C
The remaining stray product had a slightly tricky secondary structure at the 3' end. It will be run at a series of annealing temperatures in a PCR machine capable of a temperature gradient.
Construction of riboswitch plasmid with Gibson Assembly
- DNA from lanes 27+28, 27+29, 27+30 from gels run on Friday fused together with Gibson assembly to produce riboswitch construct without replacement of the first 8 codons of lac I with the 8 codons native to the gene downstream of the riboswitch.
- DNA from lanes 22,23 and 24 fused with riboswitch DNA produced two weeks ago to produce riboswitch construct with replacement of the first 8 codons of lac I with the 8 codons native to the gene downstream of the riboswitch.
Transformation of Bacillus with riboswitch construct
- Plasmids made by Gibson transformed into bacillus cells made two weeks ago and transformants plated out on 5μg/ml chloramphenicol plates.
Tuesday
Wednesday
Thursday
Friday
