Step 1: Design Primers
To use our primer design tool, click here.
Step 2: Run PCR Reaction
Follow the protocol for your favorite high fidelity DNA polymerase and buffer system.
Don't have a favorite? Here is ours:
- Buy this master mix.
- Aliquot master mix and freeze
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For each PCR reaction, mix:
- 8.50 uL nuclease free water
- 1.25 uL primer mix (10 µM each)
- 0.75 uL DMSO
- 2.00 uL of 2ng/uL template DNA
- 12.5 uL master mix (add last, immediately before PCR)
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Run this thermal cycler program
- 98°C for 5 minutes
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Repeat 30 times
- 98°C for 30 seconds
- Suggested annealing temp for 30 seconds
- 72°C for 2-4 minutes, depending on plasmid size
- 72°C for 5 minutes
- Hold at 4°C
Step 3: Check PCR Reaction
Run an agarose gel to confirm that you have amplified the plasmid.
Step 4: Remove template DNA
To digest methylated DNA, add 0.5 uL DPNI to your PCR reaction (see more here).
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Mix well and incubate at 37°C for 1 hour, or overnight at room temperture.
Step 5: Transform
Follow your the transformation protocol of your favorite competent cells. We transform with 2 uL.
Step 6: Pick Colonies and Sequence
Not all colonies will have the correct mutation. Pick approximately four of them, miniprep, and sequence.