Lowering linear plasmid copy-number
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This is a protocol to lower the copy number of pGKL1 for directed evolution. Created by the luilab (www.liulab.com)
Yeast strain with "Dummy" polymerase and methionine promoter (MET3) integrated.
Yeast synthetic defined media
Methionine stock solution (2mg per ml)
- In a PCR tube, mix the components on ice in the order they are listed above.
- Perform thermocycling program
- 95 °C 5 min
- 95 °C 30 s
- TH 30 s
- 72 °C 1 min for each 1 kb PCR product
- Repeat steps 2-4 a total of 12-36 times (24 is standard).
- 72 °C 5 min
- 12 °C hold
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
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Relevant papers and books
- Goldbeter, A and Koshland, DE (1981) - Proc Natl Acad Sci U S A 78(11) 6840-4 PMID 6947258
- Jacob, F and Monod, J J (1961) - Mol Biol 3(3) 318-56 PMID 13718526
- Ptashne, M (2004) Genetic Switch: Phage Lambda Revisited - Cold Spring Harbor Laboratory Press ISBN 0879697164
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