Team:British Columbia/Notebook/Week 6

From 2011.igem.org

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[[File:UbcTubesonice.jpg]]
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'''Beta-pinene''
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==July 11 2011==
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<b>beta-pinene</b>
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*Transformation plates taken out.
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*All 3 plates (P2SDM1, P2SDM2, P2SDM3) have colonies.
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*Set up overnight culture for miniprepping.
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<b>(-)-limonene</b>
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All 3 transformation plates (P2SDM1, P2SDM2, P2SDM3) have colonies! Marianne set up overnight cultures, mini-prepped them and sent them for sequencing. Hopefully the SDM worked and the beta-pinene synthase gene no longer has illegal cut sites.
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*SDM PCR has failed thus far.
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*Troubleshooting: Use less pfu as the glycerol content in the enzyme could alter the reaction - prepared 3 samples, each with 0.5uL pfu, 1ul pfu, and 2ul pfu, respectively. Also prepare a sample using 0.5 taq polymerase only. Also increased the annealing temperature and elongation time.
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==July 12 2011==
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'''(-)-limonene'''
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<b>beta-pinene</b>
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*Miniprep one colony from each of the three plates.
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<b>(-)-limonene</b>
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SDMs have failed thus far. More troubleshooting: use less pfu as the glycerol content in the enzyme could alter the reaction - prepared 3 samples, each with 0.5uL pfu, 1ul pfu, and 2ul pfu, respectively. Also prepare a sample using 0.5 taq polymerase only. Also increased the annealing temperature and elongation time.
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*Gel verification shows no bands
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*Troubleshooting: Use a different cycling condition
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Again, gel verification shows no bands. More troubleshooting: use a different cycling condition.
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<b>1,8-cineole</b>
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Again, no bands. Something is amiss...
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*After 5 SDMs, transformations, and minipreps, Jacob should now have ECORI-less, SPEI-less, XBAI-less, PSTI-less pg-tps-cin and pgxe-tps-cin. The gel of the restriction digest has the correct number of bands. Sequencing is the next step to confirm this result.
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==July 13 2011==
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'''1,8-cineole'''
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<b>beta-pinene</b>
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*sent miniprepped samples for sequencing.
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[[File:UbcTubesonice.jpg]]
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<b>(-)-limonene</b>
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After 5 SDMs, transformations and minipreps, Jacob should now have ECORI-less, SPEI-less, XBAI-less, PSTI-less pg-tps-cin and pgxe-tps-cin. The gel of the restriction digest has the correct number of bands. Sequencing is the next step to confirm this result.
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*Gel verification of SDM products shows no bands.
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Revision as of 03:27, 4 August 2011

Team: British Columbia - 2011.igem.org

'Beta-pinene

All 3 transformation plates (P2SDM1, P2SDM2, P2SDM3) have colonies! Marianne set up overnight cultures, mini-prepped them and sent them for sequencing. Hopefully the SDM worked and the beta-pinene synthase gene no longer has illegal cut sites.

(-)-limonene

SDMs have failed thus far. More troubleshooting: use less pfu as the glycerol content in the enzyme could alter the reaction - prepared 3 samples, each with 0.5uL pfu, 1ul pfu, and 2ul pfu, respectively. Also prepare a sample using 0.5 taq polymerase only. Also increased the annealing temperature and elongation time.

Again, gel verification shows no bands. More troubleshooting: use a different cycling condition.

Again, no bands. Something is amiss...

1,8-cineole

UbcTubesonice.jpg

After 5 SDMs, transformations and minipreps, Jacob should now have ECORI-less, SPEI-less, XBAI-less, PSTI-less pg-tps-cin and pgxe-tps-cin. The gel of the restriction digest has the correct number of bands. Sequencing is the next step to confirm this result.

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