Corum:Whole Plasmid PCR

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==Overview==
==Overview==
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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]. [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1147 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.
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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. Primers should be sense-antisense pairs. Optimal amplification occurs with 30-35 b primers at 0.5 μM final concentration. At higher concentrations, the primers bind to each other, inhibiting amplification. For longer primers, the annealing temperature will need to be adjusted from the one mentioned in this protocol.
==Materials==
==Materials==
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For a 50 μL WP-PCR reaction:
For a 50 μL WP-PCR reaction:
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* 36 μL H<sub>2</sub>O
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* 37 μL H<sub>2</sub>O
* 5 μL 10X PFU Ultra PCR buffer
* 5 μL 10X PFU Ultra PCR buffer
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* 5 μL 100 μM sense/antisense primer mix (10 μM final)
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* 5 μL 10 μM sense/antisense primer mix (0.5 μM final, each)
* 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]
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* 1 μL [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1147 PFU ligase]
 
==Procedure==
==Procedure==
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# In a PCR tube, mix the components on ice in the order they are listed above. Mix gently and spin.
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# 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
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## Ligation: 55 °C 30 s (time?)
 
## 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
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## Final ligation: 55 °C 1 hr
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## 12-16 °C hold
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## 12 °C hold
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# 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]
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*'''SC 18:46, 23 July 2012 (EDT)''':
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*'''SC 12:50, 13 December 2012 (EST)''':
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  

Current revision

Contents

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. Primers should be sense-antisense pairs. Optimal amplification occurs with 30-35 b primers at 0.5 μM final concentration. At higher concentrations, the primers bind to each other, inhibiting amplification. For longer primers, the annealing temperature will need to be adjusted from the one mentioned in this protocol.

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, each)
  • 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

  1. In a PCR tube, add the components on ice in the order they are listed above. Mix gently and spin.
  2. Perform the following thermocycling program:
    1. Initial melting: 95 °C 2 min
    2. Melting: 95 °C 20 s
    3. Annealing: Ta °C 20 s, where Ta = Tm - 5 °C
    4. Elongation: 72 °C 2 min / kb template
    5. Repeat steps 2-4 a total of 30 times
    6. Final elongation: 72 °C 30 min
    7. 12-16 °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.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

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

or instead, discuss this protocol.

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