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 | ## Initial melting: 95 °C 2 min | ||
## 95 °C | ## Melting: 95 °C 20 s | ||
## T<sub> | ## Annealing: T<sub>a</sub> °C = 20 s, where T<sub>a</sub> = T<sub>m</sub> - 5 °C | ||
## 72 °C 2 min | ## Elongation: 72 °C 2 min / kb template | ||
## Repeat steps 2-4 a total of 12-20 times (20 is standard). | ## Repeat steps 2-4 a total of 12-20 times (20 is standard). | ||
## 72 °C | ## Final elongation: 72 °C 30 min | ||
## 12 °C hold | ## 12 °C hold | ||
# Verify product with [http://openwetware.org/wiki/Corum:Gel_Electrophoresis gel electrophoresis]. | # Verify product with [http://openwetware.org/wiki/Corum:Gel_Electrophoresis gel electrophoresis]. | ||
Revision as of 20:10, 25 August 2012
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
- In a PCR tube, mix the components on ice in the order they are listed above.
- Perform the following thermocycling program:
- Initial melting: 95 °C 2 min
- Melting: 95 °C 20 s
- Annealing: Ta °C = 20 s, where Ta = Tm - 5 °C
- Elongation: 72 °C 2 min / kb template
- Repeat steps 2-4 a total of 12-20 times (20 is standard).
- Final elongation: 72 °C 30 min
- 12 °C hold
- Verify product with gel electrophoresis.
- Quantify product with quantifluore DNA quantification.
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.
