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Team:Cambridge/Diary/Week 9
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Andreas progressed with the PCR reactions FINISH THIS | Andreas progressed with the PCR reactions FINISH THIS | ||
| - | Stuart meticulously finalised his list for sporulation medium and aims to have the ingredients by friday. He also did a major rehall on our freezer stocks as things were getting a little unorganised. | + | Stuart meticulously finalised his list for sporulation medium and aims to have the ingredients by friday. He also did a major (really?) rehall on our freezer stocks as things were getting a little unorganised. |
Paul had lots of fun designing his own protocol for IPTG induction of ratiometrica in E.coli but has to wait a night to see his results - These are unlikely to be positive and not just because of Paul's ineptitude but mainly because we're pretty sure this isn't actually ratiometrica. This is because today Emmy also mini-prepped our ratiometrica cells and did a restriction digest - results were inconclusive but its starting to look like our two colonies were the result of a transformation of the template vector that might have got into the gel well we extracted! A more elaborate restriction digest will tell us more. Meanwhile there's no harm in attempting gibson again! | Paul had lots of fun designing his own protocol for IPTG induction of ratiometrica in E.coli but has to wait a night to see his results - These are unlikely to be positive and not just because of Paul's ineptitude but mainly because we're pretty sure this isn't actually ratiometrica. This is because today Emmy also mini-prepped our ratiometrica cells and did a restriction digest - results were inconclusive but its starting to look like our two colonies were the result of a transformation of the template vector that might have got into the gel well we extracted! A more elaborate restriction digest will tell us more. Meanwhile there's no harm in attempting gibson again! | ||
Revision as of 19:39, 22 August 2012
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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 could be run. 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 vector was difficult 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
Emmy ran gels for ratiometrica and the lux-mOrange fusion. Of these we managed to extract the smaller fragments for the fluorescent construct and got a quarter of the vector for the lux construct... slow progress is being made! just one quarter of a vector and two more fluorescent fragments to get and we'll all be set for gibson (which always works...). As the band wasn't too clear for the lux construct Emmy has rerun the pcr for this and also tried to get the other quarter of the vector we're still missing. FILL RESULTS!
Andreas progressed with the PCR reactions FINISH THIS
Stuart meticulously finalised his list for sporulation medium and aims to have the ingredients by friday. He also did a major (really?) rehall on our freezer stocks as things were getting a little unorganised.
Paul had lots of fun designing his own protocol for IPTG induction of ratiometrica in E.coli but has to wait a night to see his results - These are unlikely to be positive and not just because of Paul's ineptitude but mainly because we're pretty sure this isn't actually ratiometrica. This is because today Emmy also mini-prepped our ratiometrica cells and did a restriction digest - results were inconclusive but its starting to look like our two colonies were the result of a transformation of the template vector that might have got into the gel well we extracted! A more elaborate restriction digest will tell us more. Meanwhile there's no harm in attempting gibson again!
Thursday
Friday
