Team:Brown/Notebook/August16

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Monday, August 16, 2010

Miniprep of CI-pGEM (from XL1B)

  • CI - 314ng/ul

Double digest of Mnt-pGEM, Lovtwo-pGEM

50ul total volume

  • 5ul NEB Buffer 3
  • 2ul DNA
  • 0.5ul 100x BSA
  • 1ul BglII
  • 1ul XhoI
  • 40.5ul dH2O

Gel of the above digest

  1. Ladder
  2. Lovtwo
  3. Mnt

Gel 81610.jpg

Re-ligations

We are using more insert because the previous ligations were not fully successful.

  1. Lovtwo into pGEM
  2. bgl-CI into pGEM
  3. bgl-AraC into pGEM
  4. bgl-Mnt into pGEM

Concentrations:

  • pGEM - 50ng/ul
  • bgl-CI - 17.3ng/ul
  • bgl-AraC - 14.2ng/ul
  • bgl-Mnt - 15ng/ul

17ul total volume:

  • 7.5ul ligase buffer
  • 1ul T4 DNA ligase
  • 0.5ul pGEM vector
  • 8ul DNA
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