Corum:Whole Plasmid PCR: Difference between revisions
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==Materials== | ==Materials== | ||
For a 50 μL | For a 50 μL WP-PCR reaction: | ||
* | * 36 μL H<sub>2</sub>O | ||
* 5 μL 10X PFU Ultra PCR buffer | * 5 μL 10X PFU Ultra PCR buffer | ||
* 5 μL 100 μM sense/antisense primer mix (10 μM final) | * 5 μL 100 μM sense/antisense primer mix (10 μM final) | ||
| Line 12: | Line 12: | ||
* 1 μL 5 nM plasmid template (0.1 nM final) | * 1 μL 5 nM plasmid template (0.1 nM final) | ||
* 1 μL [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1095 PfuUltra II fusion HS DNA polymerase] | * 1 μL [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1095 PfuUltra II fusion HS DNA polymerase] | ||
* 1 μL [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1147 PFU ligase] | |||
==Procedure== | ==Procedure== | ||
| Line 18: | Line 19: | ||
## Initial melting: 95 °C 2 min | ## Initial melting: 95 °C 2 min | ||
## Melting: 95 °C 20 s | ## Melting: 95 °C 20 s | ||
## Annealing: T<sub>a</sub> °C | ## Annealing: T<sub>a</sub> °C 20 s, where T<sub>a</sub> = T<sub>m</sub> - 5 °C | ||
## Elongation: 72 °C 2 min / kb template | ## Elongation: 72 °C 2 min / kb template | ||
## Ligation: 55 °C 30 s (time?) | ## Ligation: 55 °C 30 s (time?) | ||
## Repeat steps 2-4 a total of | ## Repeat steps 2-4 a total of 30 times | ||
## Final elongation: 72 °C 30 min | ## Final elongation: 72 °C 30 min | ||
## Final ligation: 55 °C 1 hr | ## Final ligation: 55 °C 1 hr | ||
Revision as of 12:53, 4 September 2012
Overview
Whole plasmid polymerase chain reaction (WP-PCR) protocol using PfuUltra II fusion HS DNA polymerase. PFU ligase is used in parallel to repair the nicked plasmid products as PCR proceeds. It is imperative to use 5'-phosphorylated primers for the ligase to be able to repair the nicks.
Materials
For a 50 μL WP-PCR reaction:
- 36 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 PfuUltra II fusion HS DNA polymerase
- 1 μL PFU ligase
Procedure
- In a PCR tube, mix the components on ice in the order they are listed above. Mix gently and spin.
- 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
- Ligation: 55 °C 30 s (time?)
- Repeat steps 2-4 a total of 30 times
- Final elongation: 72 °C 30 min
- Final ligation: 55 °C 1 hr
- 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.
