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Team:Cambridge/Diary/Week 9
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Ratiometrica still not showing any YFP fluorescence (CFP hard to tell) but some of the restreaked cultures have now been place in amp resistant liquid medium for growth in preparation for IPTG induction. Some cultures were also prepared for a restriction digest tomorrow! | Ratiometrica still not showing any YFP fluorescence (CFP hard to tell) but some of the restreaked cultures have now been place in amp resistant liquid medium for growth in preparation for IPTG induction. Some cultures were also prepared for a restriction digest tomorrow! | ||
| - | Emmy ran a positive control on the PCR machine with an increased loading dye concentration and despite producing the weirdest gel I've ever seen, did locate the band we wanted! PCR back on track! In light of this, Stuart and Andreas | + | Emmy ran a positive control on the PCR machine with an increased loading dye concentration and despite producing the weirdest gel I've ever seen, did locate the band we wanted! PCR back on track! In light of this, Stuart and Andreas started a PCR on some B. subtilis, the fluoride riboswitch template and the Magnesium riboswitch template. Unfortunately they only discovered today that they require gibson primers for the linearised standard vector for the registry so the entire construct will be a little delayed. |
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| + | PCR results: Due to lack of primers only the colony PCR and the two halves of the Mg RS vector. Only the smaller fragment of the vector showed on the gel! After diagnosis we concluded that we'd used completely the wrong protocol for colony PCR and that it was a 5.5kb fragment that didn't run on the gel for the Mg RS vectorso difficult to get PCR conditions right. | ||
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| + | We also discovered we'd been using 10 times as much primer as we needed in all our PCR reactions which resulted in a severe lack of primers today! more delay as we order more! | ||
===Wednesday=== | ===Wednesday=== | ||
Revision as of 11:52, 22 August 2012
| Week: | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
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Contents |
Monday
Tested the Ratiometrica cultures for YFP fluorescence and none visible yet... However, we remain optimistic and our current theory is that the YFP we are using produces a very poor signal. Also, there may have been some CFP fluorescence (Although this is hard to tell as there is some autofluorescence of the cells under this excitation) meaning we might have put our proteins int he wrong way around (which doesn't matter too much thankfully). In order to know for sure whats going on we restreaked one of our colonies to grow up over night in preparation for a restriction digest and IPTG induction test.
Meanwhile Andreas and Stuart started deciphering Jolyon and Oli's information for turning the riboswitches into BioBricks. Andreas also continued work on the Arduino software. Paul and Andreas also started construction of the cuvette holders for the hardware part of the instrumentation.
Tuesday
Mylar reflective film has arrived and the first cuvette holder has been completed!
Ratiometrica still not showing any YFP fluorescence (CFP hard to tell) but some of the restreaked cultures have now been place in amp resistant liquid medium for growth in preparation for IPTG induction. Some cultures were also prepared for a restriction digest tomorrow!
Emmy ran a positive control on the PCR machine with an increased loading dye concentration and despite producing the weirdest gel I've ever seen, did locate the band we wanted! PCR back on track! In light of this, Stuart and Andreas started a PCR on some B. subtilis, the fluoride riboswitch template and the Magnesium riboswitch template. Unfortunately they only discovered today that they require gibson primers for the linearised standard vector for the registry so the entire construct will be a little delayed.
PCR results: Due to lack of primers only the colony PCR and the two halves of the Mg RS vector. Only the smaller fragment of the vector showed on the gel! After diagnosis we concluded that we'd used completely the wrong protocol for colony PCR and that it was a 5.5kb fragment that didn't run on the gel for the Mg RS vectorso difficult to get PCR conditions right.
We also discovered we'd been using 10 times as much primer as we needed in all our PCR reactions which resulted in a severe lack of primers today! more delay as we order more!
Wednesday
Thursday
Friday
