Jessica Karen Wong/Notebook/2007-6-29

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Contents

To Do

  • Digest E0240 with Mfe1 and Nsi1
  • PCR Clean E0240
  • Ligate E and 3K3, transform?
  • Colony PCR green T, and redo blue C gel
  • Miniprep B0032?

Overnights

  • B0032 has good growth
  • Also saw colonies on green T (promoter tester on Tet)

Digest

  • NEB recommends not double digesting Mfe1 and Nsi1
  • Doing a sequential digest Mfe1 then Nsi1
  • used 3ul of the scarred E0240 in the Mfe1 digest and all of the digested product in the 2nd (Nsi) digest
  • Mfe1 requires buffer 4 and Nsi1 requires buffer 3
  • Did a PCR cleanup in between the two digests

Colony PCR

  • Did a colony PCR on the 2 green (promoter tester) T colonies
  • Redid the colony PCR on the 7 blue (RBS tester) C colonies
  • Used VF and VR 40 uM primers
  • Made new 2.5uM dNTP's
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