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Site Directed Mutagenesis with Pfu Turbo
Author: Gozani Lab, updated date: , view: 9, Q&A: 0
Tags: PCR, Pfu Turbo and Site Directed Mutagenesis

Materials and Reagents

PCR supplies

Pfu Turbo (Stratagene, now Agilent)

Equipment

PCR machine

PCR tubes

PCR tube rack

Procedure

PCR  

ComponentVolume (μL)
dd H2O33.5
10X Pfu Turbo Reaction mix buffer5
2.5 mM dNTPs
2
DMSO2.5
Primer 1 (Final [ ] should be 0.4 μM)2
Primer 2 (Final [ ] should be 0.4 μM)
2
Template DNA (10pg-200ng)2
Pfu Turbo polymerase1


  1. Add the components into a PCR tube

    1. Don't forget to perform a control reaction without primers

  2. Run the following PCR protocol using a PCR machine

    Step 1Heat 95C for 2min
    Step 2
    Heat 95C for 20s
    Step 3
    Annealing 55C for 30s
    Step 4
    Elongation 68C for 14 min
    (depends on size of plasmid)
    Step 5
    Repeat steps 2-4 for 19 cycles 
    Step 6
    Elongation 68C for  10 min
    Step 7
    4C forever
    Step 8
    End
  3. After PCR has run transfer 30uL to a fresh microfuge tube

  4. Add 1uL of DpnI

  5. Incubate at 37C for 1 hr.

  6. During this incubation take 1uL of the remaining 20uL of PCR to run on an agarose gel.

  7. Take 2uL of DpnI-digested mixture and transform into bacteria (don't forget to transform the control reaction that did not have primers).

References

This protocol is adapted from the PCR PROTOCOL FOR PfuTURBO DNA POLYMERASE by Stratagene (early 2000's). 

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