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Edinburgh/18 July 2008
From 2008.igem.org
Week 5
Thursday 17 July 08
- 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).
- 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.
- 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)
- pSB1A2+appY cultures set up in beaker for maxiprep tomorrow (X3). (AM)
