Lowering linear plasmid copy-number

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Current revision (16:58, 4 September 2013) (view source)
(Materials)
 
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==Materials==
==Materials==
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For a 50 μL PCR reaction:
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Yeast strain with "Dummy" polymerase and methionine promoter (MET3) integrated.
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* 35 μL H<sub>2</sub>O
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Yeast synthetic defined media
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* 5 μL 10X PCR buffer
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* 5 μL 2mM dNTPs (each)
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Methionine stock solution (2mg per ml)
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* 1.5 μL 50mM MgCl<sub>2</sub>
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* 1 μL 50μM sense primer
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* 1 μL 50μM antisense primer
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* 1 μL 5nM DNA template
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* 0.5 μL TAQ DNA polyermerase
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==Procedure==
==Procedure==

Current revision

Contents

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. 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

Notes

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.

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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

Contact

  • Who has experience with this protocol?

or instead, discuss this protocol.


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