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Edinburgh/30 June 2008
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Week 3
Monday 30 June 08
- Plasmid DNA minipreps:
M1 to M6: pSB1A2+dxs white colonies
M7: Babel1+glgC white colony
M8 to M12: Babel2+glgC white and pale blue colonies
Minipreps M1 to M6 were digested with EcoRI and PstI (Gel 2). The expected pattern is pSB1A2 vector band at 2.04kb (2079 bp less prefix and suffix) and dxs insert band around 1.9kb. M1 showed bands around 1.1kb and 2.1kb. M5 showed no DNA. The other four showed a vector-like band around 2kb and a fainter, fuzzier band around 3kb, possibly a 'ghost' band. Thus none of the clones show the expected pattern of bands (although it is conceivable, since the vector and insert bands are so close in size, that they may be lying on top of each other; dxs has an internal EcoRV site at +504 and two HindIII sites at +606 and +1230, whereas pSB1A2 lacks such sites so this could be used to check). In conjunction with the total lack of growth on the appY plates, this suggests that something went wrong in the cloning procedure. The next step is to run the remaining ligation material on a gel and see what it looks like.
