PCR Mutagenesis (QuikChange® II)

Link to full manual

1. Primer Creation:

• Find the region of DNA you want to do mutagenesis on. Selecting that region in a GUI application, highlight an additional 10-15 bases on each end of the starting selection. If possible, try to make each end terminate on at least 1 to 2 bases of GC rich areas.
• If your’re doing a non-deletion or non-insertion mutagenesis, use the following equation to find your melting temp. TM = 81.5 + 0.41(%GC) - (675/length) - %mismatch; where: length is the length of your primer, %GC is the GC content in percent form (ex: 52.2% ~ 52.2), and %mismatch is the number of mutated bases divided by the length of the primer in percent form like the GC content.
• If your doing insertions or deletions, use this equation instead: TM = 81.5 + 0.41(%GC)- (675/length); where: length is the length of your primer not including the bases are your inserting or deleting, %GC is the GC content in percent form (ex: 52.2% ~ 52.2).
• You may want to check additional problems with your primers like the formation of secondary structures. The TM needs to be greater than or equal to 78°C.

2. Prepare the reaction as indicated below:

• 5 µl of 10× reaction buffer
• X µl (10 ng) of 4.5-kb plasmid
• X µl (125 ng) of oligonucleotide primer #1
• X µl (125 ng) of oligonucleotide primer #2
• 1 µl of dNTP mix
• 3 µl of QuikSolution reagent
• X µl ddH2O to a final volume of 50 µl
• 1 µl of PfuUltra HF DNA polymerase (2.5 U/µl) to a final volume of 51 µl

3. Cycle each reaction using the cycling parameters outlined in Table I.

Segment	Cycles	Temperature	Time
1	1	95°C	1 minute
2	18	95°C	50 seconds
		60°C	50 seconds
		68°C	1 min/kb of plasmid
3	1	68°C	7 minutes

4. Following temperature cycling, place the reaction tubes on ice for 2 minutes to cool the reactions to ≤37°C.

5. Add 1 µl of the Dpn I restriction enzyme (10 U/µl) directly to each amplification reaction. Gently and thoroughly mix by pipetting.

6. Spin down the reaction mixtures in a microcentrifuge for 1 minute, then immediately incubate the reactions at 37°C for 1 hour to digest.