User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/03/07

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Split PCR for M8S Mutation

Using a modified version of 2-stage QuikChange the M8S mutation should be inserted into the plasmid which already has the M33S and M103S mutations.

Protocol

First Stage

1st stage
Tube sterile H2O Pfu Buffer M33S/M103S mutated hemoglobin For primer (M8S f) Rev primer (M8S r)dNTPs Pfu Turbo wax
(10X) (~100 ng/μL) (12.5 ng/μL) (12.5 ng/μL) (10 mM ea) (2.5 U/μL)
Experimental-forward 27 μL 5 μL 5 μL 10 μL 0 μL 2 μL 1 μL 50 μL
Experimental-reverse 27 μL 5 μL 5 μL 0 μL 10 μL 2 μL 1 μL 50 μL
(-) Control-forward 28 μL 5 μL 5 μL 10 μL 0 μL 2 μL 0 μL 50 μL
(-) Control-reverse 28 μL 5 μL 5 μL 0 μL 10 μL 2 μL 0 μL 50 μL
  • Combine all components of each mixture in sterile PCR tubes, except for Pfu Turbo and wax, mix well, but gently
  • Add Pfu Turbo and mix gently with pipet
  • Add wax on top of mixture and place on thermoclycler
  • 30" @ 95°C
  • Cycle through the following six times:
      1. 30" @ 95°C
      2. 1' @ 55°C
      3. 5' @ 68°C
  • Remove from thermocycler, and pipet wax off of each reaction mixture
  • Begin second stage

Second Stage

2nd stage
Tube 1st stage-forward 1st stage-reverse Pfu Turbo wax
(2.5 U/μL)
Experimental 25 μL 25 μL 1 μL 50 μL
(-) Control 25 μL 25 μL 0 μL 50 μL
  • Combine mixtures from first stage and mix together gently with pipet, then add Pfu Turbo and mix again gently with pipet
  • Add wax on top of mixture and place on thermoclycler
  • 30" @ 95°C
  • Cycle through the following 20 times:
      1. 30" @ 95°C
      2. 1' @ 55°C
      3. 5' @ 68°C
  • 15' @ 68°C
  • hold @ 4°C


Digestion of Original DNA

  • Remove the wax from both tubes.
  • Add 1μL of DpnI to both PCR tubes.
  • Incubate the tubes on the heatblock at 37°C for one hour.
  • Remove and store at -20°C



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