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Team:Cambridge/Diary/Week 12
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===Monday=== | ===Monday=== | ||
| - | PCR of the biobrick backbone is still proving troublesome, as no bands are coming out. Oli's trying the PCRs at ever higher | + | PCR of the biobrick backbone is still proving troublesome, as no bands are coming out. Oli's trying the PCRs at ever higher temperatures on the backbone, but we've realised that there's a CG palindrome in both the prefix and suffix sequence, which may be causing our primers to self-anneal and form primer dimers. If these continue to fail, we'll probably have to switch to traditional ligation. |
But at least both a restriction digest and a colony PCR confirmed the identity of our Magnesium Riboswitch construct. One of the colonies seems to be fluorescing far more green than the other, which may be down to a mutation that has upregulated our sfGFP. We'll need to do some characterization before we can confirm that, though. | But at least both a restriction digest and a colony PCR confirmed the identity of our Magnesium Riboswitch construct. One of the colonies seems to be fluorescing far more green than the other, which may be down to a mutation that has upregulated our sfGFP. We'll need to do some characterization before we can confirm that, though. | ||
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| + | We've also started producting spores, with some different ''bacillus'' strains cultured up in sporulation medium. Who would have thought that inducing these bacteria to form what they should do naturally would be so complicated... | ||
===Tuesday=== | ===Tuesday=== | ||
Revision as of 01:12, 16 September 2012
| Week: | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 |
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Contents |
Monday
PCR of the biobrick backbone is still proving troublesome, as no bands are coming out. Oli's trying the PCRs at ever higher temperatures on the backbone, but we've realised that there's a CG palindrome in both the prefix and suffix sequence, which may be causing our primers to self-anneal and form primer dimers. If these continue to fail, we'll probably have to switch to traditional ligation.
But at least both a restriction digest and a colony PCR confirmed the identity of our Magnesium Riboswitch construct. One of the colonies seems to be fluorescing far more green than the other, which may be down to a mutation that has upregulated our sfGFP. We'll need to do some characterization before we can confirm that, though.
We've also started producting spores, with some different bacillus strains cultured up in sporulation medium. Who would have thought that inducing these bacteria to form what they should do naturally would be so complicated...
Tuesday
Wednesday
Thursday
Jolyon and Andreas came back today, allowing us to share the workload between plenty of people again. On top of that, the construct from DNA 2.0 turned up, along with a load of high quality plate reader plates.
Friday
Analysis of the sequence sent to us of the biobricks we had hoped to make indicate that the riboswitches have inserted - backwards. The current sequence is Backbone-Front of Suffix-End of Prefix-Riboswitch-End of Suffix-Start of Prefix. This almost success is somewhat mocking. However, Jolyon is starting to try to assemble our biobricks with standard assembly instead - hopefully less can go wrong with this. It's a shame to give up on Gibson, but it has been a cruel master this Summer.
Andreas and Paul started getting some images of the spores we made over the last few days. We should be able to use this microscopy as an assay when we are testing the efficacy of the new promotor we're hoping to submit. Speed of germination should be fairly simple to measure with this capacity.
