User:Cassandra M Barrett/Notebook/Open Chromatin/2015/09/26

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LCR Transformations and gel results

Purpose: to test effectiveness of LCR by transforming E. coli with ligated DNA

Methods:

Reactions:

  • Reaction 1
  • Reaction 10
  • Reaction 11
  • MV10 uncut
  • MV10 XbaI/MBN/PNK
  • MV10 XbaI/MBN/ not PNK
  • Water

Follow standard DH5alpha transformation protocol:

  • Add 50uL of DH5alpha turbo cells to each tube with appropriate sample (2.5uL of each)
  • Incubate on ice for 30min
  • Heat shock at 42C for 35sec
  • Incubate on ice for 2min
  • Add 750uL of SOC media (RT) to each tube
  • Shake 37C for 1hr
  • Plate on LB+Amp (Spin down, remove 200uL of supernatant, resuspend and plate 100uL)
  • Incubate overnight at 37C

Run 2uL of reactions 1-11 on gel


Image:LCRPCR.jpg

We see vague larger bands on 10 and 11, probably just reannealed MV10 No sample in rxn 8 tube for some mysterious reason....


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