Corum:Whole Plasmid PCR: Difference between revisions
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# In a PCR tube, mix the components on ice in the order they are listed above. | # In a PCR tube, mix the components on ice in the order they are listed above. | ||
# Perform the following thermocycling program: | # Perform the following thermocycling program: | ||
## 95 °C 1 min | ## 95 °C 1 min (initial melting) | ||
## 95 °C 30 s | ## 95 °C 30 s (melting) | ||
## T<sub>H</sub> = 55 °C | ## T<sub>H</sub> = 55 °C 60 s (annealing, ligation) | ||
## 72 °C 2 min for each 1 kb PCR product | ## 72 °C 2 min for each 1 kb PCR product (elongation) | ||
## Repeat steps 2-4 a total of 12- | ## Repeat steps 2-4 a total of 12-20 times (20 is standard). | ||
## 72 °C 20 min | ## 72 °C 20 min (final elongation) | ||
## 55 °C 60 min (final ligation) | |||
## 12 °C hold | ## 12 °C hold | ||
Revision as of 17:39, 24 July 2012
Overview
Whole plasmid PCR protocol using PFU Ultra DNA polymerase.
Materials
For a 50 μL whole plasmid PCR PCR reaction:
- 35 μL H2O
- 5 μL 10X PFU Ultra PCR buffer
- 5 μL 2mM (each) dNTP mix
- 1 μL 5nM plasmid template (0.1 nM final)
- 1 μL 10μM sense primer (200nM final)
- 1 μL 10μM antisense primer (200nM final)
- 1 μL PFU Ultra DNA polyermerase
- 1 μL PFU ligase
Procedure
- In a PCR tube, mix the components on ice in the order they are listed above.
- Perform the following thermocycling program:
- 95 °C 1 min (initial melting)
- 95 °C 30 s (melting)
- TH = 55 °C 60 s (annealing, ligation)
- 72 °C 2 min for each 1 kb PCR product (elongation)
- Repeat steps 2-4 a total of 12-20 times (20 is standard).
- 72 °C 20 min (final elongation)
- 55 °C 60 min (final ligation)
- 12 °C hold
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- 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.
References
Relevant papers and books
- Sambrook, J and Russell, DW (2001) Molecular Cloning: A Laboratory Manual (Volume II) - Cold Spring Harbor Laboratory Press ISBN 0879695773
Contact
- Sean P Corum
- SC 18:46, 23 July 2012 (EDT):
or instead, discuss this protocol.
