Edinburgh/18 July 2008

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=== Week 5 ===
=== Week 5 ===
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==== Thursday 17 July 08 ====
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==== Friday 18 July 08 ====
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* PCR for ''cenA'', ''cenB'', ''cenC'' and ''cex'' ('''P16''', '''P17''', '''P18''', '''P19'''). Annealing 58°C, extension 115s (''cenB'' and ''cenC'' are both over 3kb) and included 5% v/v glycerol in the reactions since in the past that has been helpful with high GC templates. PCR products purified to be run on gel tomorrow (against wisdom, since they should have been run on gel both before and after purification).
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* Made pSB1A2+''appY'' Maxiprep *X3*. (AM)
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* Results of transformations: plenty of colonies for ''glgC'' mutation (mutating site 1 in the mutant which already has site 2 mutated). Subbed some of these to '''plates 42, 44 and 45''' (poor communications). Only a few colonies for rbs+''dxs'', and only one of these was white. Subbed this to '''plate 43'''. Apparently there may have been some problem with the purification of this digest - may need to repeat.
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* Purified PCR products for ''cenA'', ''cenB'', ''cenC'' and ''cex'' ('''P16''', '''P17''', '''P18''', '''P19''') run on '''Gel 16'''. No visible bands. (AM)
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* Re-digested M32, M36 (''pSB1A2-crtB''), M39 and M40 (''pSB1A2-crtI'') and ran on '''Gel 15'''. Gel unsuccessful - stain smeared over 0.5-2kb region, exactly where ''crtB'' and ''crtI'' bands are expected to appear. Perhaps too much loading buffer or problem with the tank (tank with 'loose wire' was used). (AM)
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* Second attempt at PCR for ''cenA'', ''cenB'', ''cenC'' and ''cex'' ('''P20''', '''P21''', '''P22''', '''P23'''). Annealing 55°C, extension 85s; did ''not'' include glycerol. Ran unpurified PCR products on '''Gel 17''' - no bands apart from thick bands in the <0.5kb region = primers. Hence, PCR was unsuccessful. (AM)
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* pSB1A2+''appY'' cultures set up in beaker for maxiprep tomorrow ('''X3'''). (AM)
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Revision as of 11:15, 20 August 2008

Edinburgh iGEM 2008

 

Week 5

Friday 18 July 08

  • Made pSB1A2+appY Maxiprep *X3*. (AM)
  • Purified PCR products for cenA, cenB, cenC and cex (P16, P17, P18, P19) run on Gel 16. No visible bands. (AM)
  • Second attempt at PCR for cenA, cenB, cenC and cex (P20, P21, P22, P23). Annealing 55°C, extension 85s; did not include glycerol. Ran unpurified PCR products on Gel 17 - no bands apart from thick bands in the <0.5kb region = primers. Hence, PCR was unsuccessful. (AM)
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