Corum:Whole Plasmid PCR: Difference between revisions

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==Overview==
==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.
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.


==Materials==
==Materials==


For a 50 μL whole plasmid PCR PCR reaction:
For a 50 μL WP-PCR reaction:


* 35 μL H<sub>2</sub>O
* 36 μL H<sub>2</sub>O
* 5 μL 10X PFU Ultra PCR buffer
* 5 μL 10X PFU Ultra PCR buffer
* 5 μL 2mM (each) dNTP mix
* 5 μL 100 μM sense/antisense primer mix (10 μM final)
* 1 μL 5nM plasmid template (0.1 nM final)
* 1 μL 12.5 mM (each) dNTP mix (0.25 mM final)
* 1 μL 10μM sense primer (200nM final)
* 1 μL 5 nM plasmid template (0.1 nM final)
* 1 μL 10μM antisense primer (200nM final)
* 1 μL [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1095 PfuUltra II fusion HS DNA polymerase]
* 1 μL PFU Ultra DNA polyermerase
* 1 μL [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1147 PFU ligase]
* 1 μL PFU ligase


==Procedure==
==Procedure==
# 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. Mix gently and spin.
# Perform the following thermocycling program:
# Perform the following thermocycling program:
## 95 °C 1 min (initial melting)
## Initial melting: 95 °C 2 min
## 95 °C 30 s (melting)
## Melting: 95 °C 20 s
## T<sub>H</sub> = 55 °C 60 s (annealing, ligation)
## Annealing: T<sub>a</sub> °C 20 s, where T<sub>a</sub> = T<sub>m</sub> - 5 °C
## 72 °C 2 min for each 1 kb PCR product (elongation)
## Elongation: 72 °C 2 min / kb template
## Repeat steps 2-4 a total of 12-20 times (20 is standard).
## Ligation: 55 °C 30 s (time?)
## 72 °C 20 min (final elongation)
## Repeat steps 2-4 a total of 30 times
## 55 °C 60 min (final ligation)
## Final elongation: 72 °C 30 min
## Final ligation: 55 °C 1 hr
## 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 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

  1. In a PCR tube, mix 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. Ligation: 55 °C 30 s (time?)
    6. Repeat steps 2-4 a total of 30 times
    7. Final elongation: 72 °C 30 min
    8. Final ligation: 55 °C 1 hr
    9. 12 °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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