Repeat Midipreps to select new clones for sequencing for failed sequences from Oct. 2

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  • Participants: Johan, Aaron, Rebecca, Rachel, Allen, Lafia, Menakshi (Aaron did the protocol with some help from Johan and Allen)
  • Date: 10/3/2014
  • Location: BioCurious
  • Aims: Midiprep colonies from P.FAKS.bovAlphaS1.S (1), P.FAKS.bovAlphaS2(Kex+).S (3), P.FAKS.bov.Beta(A2).S (6)
    • (We did not get to midiprep P.FAKS.humkappa(kex-).S (5) tonight because we had to duplicate the glycerol stock)
  • Materials and Methods*
    • We used the Zyppy Plasmid Midiprep Kit (Protocol = Media:D4025i.pdf‎)
      • Changes to protocol: Step 9: centrifuge 5 min the 2nd time, not 1 min
    • Cultures were grown in 6 ml culture with 6 microliters 50mg/ml carbenicillen from Teknova; they had been innoculated with with single colonies from 9/12/2014 transformation plates; plastic loops (not individually wrapped but from a new bag) were used; cultures were incubated for 24 hours
    • Each was done twice to increase the chances of getting the correct sequence because Electra negative control plates had colonies on them, suggesting possible contamination