Site Directed Mutagenesis with Pfu Turbo

PCR supplies

Pfu Turbo (Stratagene, now Agilent)

PCR machine

PCR tubes

PCR tube rack

PCR  

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 1	Heat 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).
   

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