Corum:Whole Plasmid PCR: Difference between revisions
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==Overview== | ==Overview== | ||
Whole plasmid polymerase chain reaction (WP-PCR) protocol using [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1095 PfuUltra II fusion HS DNA polymerase] | Whole plasmid polymerase chain reaction (WP-PCR) protocol using [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1095 PfuUltra II fusion HS DNA polymerase]. It is imperative to use 5'-phosphorylated primers if the nicked DNA is going to be repaired downstream with ligase. PCR should be limited to templates of about 5 kb, but the protocol could probably be pushed up to 10 kb. See the PfuUltra II fusion manual. | ||
==Materials== | ==Materials== | ||
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For a 50 μL WP-PCR reaction: | For a 50 μL WP-PCR reaction: | ||
* | * 37 μL H<sub>2</sub>O | ||
* 5 μL 10X PFU Ultra PCR buffer | * 5 μL 10X PFU Ultra PCR buffer | ||
* 5 μL | * 5 μL 10 μM sense/antisense primer mix (0.5 μM final) | ||
* 1 μL 12.5 mM (each) dNTP mix (0.25 mM 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 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] | ||
==Procedure== | ==Procedure== | ||
# In a PCR tube, | # In a PCR tube, add the components on ice in the order they are listed above. Mix gently and spin. | ||
# Perform the following thermocycling program: | # Perform the following thermocycling program: | ||
## Initial melting: 95 °C 2 min | ## Initial melting: 95 °C 2 min | ||
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## Annealing: T<sub>a</sub> °C 20 s, where T<sub>a</sub> = T<sub>m</sub> - 5 °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 | ||
## Repeat steps 2-4 a total of 30 times | ## Repeat steps 2-4 a total of 30 times | ||
## Final elongation: 72 °C 30 min | ## Final elongation: 72 °C 30 min | ||
## 12-16 °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]. | ||
# Quantify product with [http://openwetware.org/wiki/Corum:DNA_Quantification quantifluore DNA quantification]. | # Quantify product with [http://openwetware.org/wiki/Corum:DNA_Quantification quantifluore DNA quantification]. | ||
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==Contact== | ==Contact== | ||
* [http://openwetware.org/wiki/User:Sean_P_Corum Sean P Corum] | * [http://openwetware.org/wiki/User:Sean_P_Corum Sean P Corum] | ||
*'''SC | *'''SC 12:50, 13 December 2012 (EST)''': | ||
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]]. | or instead, [[Talk:{{PAGENAME}}|discuss this protocol]]. |
Revision as of 10:50, 13 December 2012
Overview
Whole plasmid polymerase chain reaction (WP-PCR) protocol using PfuUltra II fusion HS DNA polymerase. It is imperative to use 5'-phosphorylated primers if the nicked DNA is going to be repaired downstream with ligase. PCR should be limited to templates of about 5 kb, but the protocol could probably be pushed up to 10 kb. See the PfuUltra II fusion manual.
Materials
For a 50 μL WP-PCR reaction:
- 37 μL H2O
- 5 μL 10X PFU Ultra PCR buffer
- 5 μL 10 μM sense/antisense primer mix (0.5 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
Procedure
- In a PCR tube, add 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
- Repeat steps 2-4 a total of 30 times
- Final elongation: 72 °C 30 min
- 12-16 °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 12:50, 13 December 2012 (EST):
or instead, discuss this protocol.