Corum:Whole Plasmid PCR

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For a 50 μL whole plasmid PCR PCR reaction:
For a 50 μL whole plasmid PCR PCR reaction:
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* 35 μL H<sub>2</sub>O
+
* 37 μL H<sub>2</sub>O
* 5 μL 10X PFU Ultra PCR buffer
* 5 μL 10X PFU Ultra PCR buffer
-
* 5 μL 2mM (each) dNTP mix
+
* 5 μL 100 μM sense/antisense primer mix (10 μM final)
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* 1 μL 5nM plasmid template (0.1 nM final)
+
* 1 μL 12.5 mM (each) dNTP mix (0.25 mM final)
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* 1 μL 10μM sense primer (200nM final)
+
* 1 μL 5 nM plasmid template (0.1 nM final)
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* 1 μL 10μM antisense primer (200nM final)
+
* 1 μL PFU Ultra II high-fidelity DNA polyermerase
* 1 μL PFU Ultra II high-fidelity DNA polyermerase
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* 1 μL PFU ligase
 
==Procedure==
==Procedure==

Revision as of 23:07, 25 August 2012

Contents

Overview

Whole plasmid PCR protocol using PFU Ultra DNA polymerase. PFU ligase is used in the reaction to repair the nicked plasmid products as PCR proceeds. It is imperative to use 5'-phosphorylated primers for the ligase to repair the nicks.

Materials

For a 50 μL whole plasmid PCR PCR reaction:

  • 37 μL H2O
  • 5 μL 10X PFU Ultra PCR buffer
  • 5 μL 100 μM sense/antisense primer mix (10 μM final)
  • 1 μL 12.5 mM (each) dNTP mix (0.25 mM final)
  • 1 μL 5 nM plasmid template (0.1 nM final)
  • 1 μL PFU Ultra II high-fidelity DNA polyermerase

Procedure

  1. In a PCR tube, mix the components on ice in the order they are listed above.
  2. Perform the following thermocycling program:
    1. 95 °C 1 min (initial melting)
    2. 95 °C 30 s (melting)
    3. TH = 55 °C 60 s (annealing, ligation)
    4. 72 °C 2 min for each 1 kb PCR product (elongation)
    5. Repeat steps 2-4 a total of 12-20 times (20 is standard).
    6. 72 °C 20 min (final elongation)
    7. 55 °C 60 min (final ligation)
    8. 12 °C hold
  3. Verify product with gel electrophoresis.
  4. Quantify product with quantifluore DNA quantification.

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. Sambrook, J and Russell, DW (2001) Molecular Cloning: A Laboratory Manual (Volume II) - Cold Spring Harbor Laboratory Press ISBN 0879695773

Contact

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