Difference between revisions of "Lowering linear plasmid copy-number"

From OpenWetWare
Jump to: navigation, search
Line 6: Line 6:
For a 50 μL PCR reaction:
Yeast strain with "Dummy" polymerase and methionine promoter (MET3) integrated.
* 35 μL H<sub>2</sub>O
Yeast synthetic defined media
* 5 μL 10X PCR buffer
* 5 μL 2mM dNTPs (each)
Methionine stock solution (2mg per ml)
* 1.5 μL 50mM MgCl<sub>2</sub>
* 1 μL 50μM sense primer
* 1 μL 50μM antisense primer
* 1 μL 5nM DNA template
* 0.5 μL TAQ DNA polyermerase

Latest revision as of 13:58, 4 September 2013


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)


  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. TH 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


Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.


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


  • Who has experience with this protocol?

or instead, discuss this protocol.