Team:Brown/Notebook/October4

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Monday, October 4, 2010

Digest and ligation of LacI into pSB1C3

LacI (linear, blunt ends) will be double digested with PstI and EcoRI and inserted into pSB1C3.

Digest

50ul total volume

  • 25ul dH2O
  • 5ul 10x buffer H
  • 2.5ul EcoRI
  • 2.5ul PstI
  • 15ul DNA

Ran 2 tubes in parallel. the dH2O used for the first tube had a plastic-y smell, so the tube was discarded and this was noted in case of later error

Cleanup

Followed Qiagen PCR cleanup protocol

Ligation

30ul total volume

  • 15ul 2x rapid buffer
  • 2ul ligase (T4)
  • 1ul vector
  • 10ul insert (LacI)
  • 2ul H2O
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