Site-Directed Mutagenesis with BioRAD iPROOF PCR kit

This protocol closely follows the Stratagene QuikChange Site-Directed Mutagenesis kit, but uses the enzymes and buffers from the BioRAD iPROOF PCR kit.

1. Prepare the reaction mixtures:

Per 50 µL PCR reaction:
dNTP	4 µL
iProof HF Buffer	10 µL
MgCl2	1.5 µL
Primer, each	? µL (125 ng)
dsDNA template	? µL (5-50 ng)
iProof enzyme	0.5 µL
dH2O	to 50 µL

I would suggest trying 5 ng of dsDNA template, as I had the highest mutation success rate at this concentration. This observation, however, has not been confirmed and is more anecdotal

2. Thermocycle the reaction according to the standard iPROOF cycling conditions, adjusting the extension time (X:XX) for the expected product length.

1.	Hot start	98ºC	1:00
2.	Denaturation	98ºC	0:10
3.	Anneal	55ºC	0:30
4.	Extension	72ºC	X:XX
5.	GOTO 2 for 12-18 cycles

The QuikChange protocol suggests the following number of cycles

Type of mutation	Number of cycles
Point mutations	12
Single amino acid changes	16
Multiple amino acid deletions or insertions	18

3. Check for amplification on agarose gel

I have had positive transformations even without positive band, so it's worth a shot to transform the product regardless of the outcome.

4. Perform DpnI digestion of amplification products to digest the parental DNA (CRITICAL!!!)

5. Add 10 U of DpnI to each reaction and incubate at 37 ºC

6. Transform 1 µL of PCR product. I did not do any PCR purification before this step

Team:British Columbia/Protocols/Site Directed Mutagenesis

From 2012.igem.org

Site-Directed Mutagenesis with BioRAD iPROOF PCR kit