Jessica Karen Wong/Notebook/2007-6-29
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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
