Lowering linear plasmid copy-number

Overview

This is a protocol to lower the copy number of pGKL1 for directed evolution. Created by the luilab (www.liulab.com)

Materials

Yeast strain with "Dummy" polymerase and methionine promoter (MET3) integrated.

Yeast synthetic defined media

Methionine stock solution (2mg per ml)

Procedure

1. In a PCR tube, mix the components on ice in the order they are listed above.
2. Perform thermocycling program
   1. 95 °C 5 min
   2. 95 °C 30 s
   3. T_H 30 s
   4. 72 °C 1 min for each 1 kb PCR product
   5. Repeat steps 2-4 a total of 12-36 times (24 is standard).
   6. 72 °C 5 min
   7. 12 °C hold

Notes

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References

Relevant papers and books

1. Goldbeter, A and Koshland, DE (1981) - Proc Natl Acad Sci U S A 78(11) 6840-4 PMID 6947258
2. Jacob, F and Monod, J J (1961) - Mol Biol 3(3) 318-56 PMID 13718526
3. Ptashne, M (2004) Genetic Switch: Phage Lambda Revisited - Cold Spring Harbor Laboratory Press ISBN 0879697164

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