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.