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Notebook: October 2011

Wednesday, October 12 2011

Over the weekend, Matt kindly made gene-specific lysis (36 uL). With Vincent's help for finding a convenient Tm calculator for single-nucleotide mutants, he made the extension PCR to add the 12 T7 promoters ahead of the lysis cassette. Vincent ran the gel for them and the results (thanks to Matt's insane Keynote skillz) are found below.

Vdog made a PCR to amplify the pSB3K1 backbone. Just so he doesn't forget, here's the protocol:

• 1 uL 10mM DNTP mix
• 10 uL buffer with MgCl2 (part of the Roche pack)
• .5 uL Polymerase (Expand Hifi regular)
• .5 uL Template (psB3K1 1:10. found in t7 promoters box -- the ligated version is in the ? box)
• 1 uL of each primer (50 uM)
• ddH20 to make 50 uL (36 uL if my count is right)

The elongation temperature was 68 C (as indicated by Roche pamphlet) for 3 minutes (for a 3 kb piece). The annealing temp is 52 C (as stated in Alessandro's sheet in the Dropbox). Vdog ran a touchdown PCR from 62 to 52 C) using 3 50 uL reactions. If all goes well, we should have 150 uL of K1 ready to go. I will run the gel tomorrow.