Lidstrom:Overlap Extension PCR

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(Sewing PCR Without Primers)
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** Use Phusion buffer & standard Phusion dNTP concentration
** Use Phusion buffer & standard Phusion dNTP concentration
** Set up an array of annealing temps and % DMSO.  
** Set up an array of annealing temps and % DMSO.  
-
*Thermocycling:
+
*Thermocycling: Use Phusion polymerase with default concentrations.
-
**?
+
#98<sup>o</sub>C, 30sec
-
 
+
#98<sup>o</sub>C, 10sec
 +
#58<sup>o</sub>C, 10sec
 +
#72<sup>o</sub>C, 30sec/kb
 +
#Repeatsteps 2-4 29x  NOTE: Justin Siegel/Janet Matsen only do 9x
 +
#72<sup>o</sub>C, 5min
 +
#4<sup>o</sub>C, forever
== Use Primers on Gel-Purified "Sewing PCR" product ==
== Use Primers on Gel-Purified "Sewing PCR" product ==
*
*

Revision as of 10:19, 2 October 2012

Back to Protocols

Janet is going to write up & illustrate some nice graphics for this! Stay tuned.

This method can be used for cloning. It can also be used to assemble neighboring fragments in Gibson into one piece.

Obtain DNA

  • PCR with primers that yield overlapping ends.
    • How much overlap?
  • Gel purify
    • Can sometimes only do a PCR cleanup if your bands are SUPER clean. You will get higher yield if you don't use a gel.

Sewing PCR Without Primers

  • use template DNA
    • Concentration?
  • Rxn concentrations
    • Use Phusion buffer & standard Phusion dNTP concentration
    • Set up an array of annealing temps and % DMSO.
  • Thermocycling: Use Phusion polymerase with default concentrations.
  1. 98o</sub>C, 30sec
  2. 98o</sub>C, 10sec
  3. 58o</sub>C, 10sec
  4. 72o</sub>C, 30sec/kb
  5. Repeatsteps 2-4 29x NOTE: Justin Siegel/Janet Matsen only do 9x
  6. 72o</sub>C, 5min
  7. 4o</sub>C, forever

Use Primers on Gel-Purified "Sewing PCR" product

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