Quikchange Kit II (PfuUltra HP DNA polymerase)
I would like to introduce point mutation in the plasmid.
Using the same kit, I have introduced point mutations in plasmid vectors as large as 15 kb.
Ok Dr Ali. I came across a publication that requires me to use more DNA template up to 75ng and 300uM of dNTP. That includes several modifications on the PCR cycling parameter.
Another one paper requires me to change the normal desalting primers to PAGE purified primers and KOD Hot Start Polymerase.
I'm quite puzzled on which to follow?
For the parameters mentioned in the first paragraph, I would rather strictly adhere to the protocol supplied with the kit. As far as primers are concerned, if a 50 mer primer is synthesized, the reaction also contains 49 mer failure, 48 mer failure, 45 mer failure etc. These failure sequences (oligos missing bases at 5’ end) may compete with full length primers in the reaction. For site-directed mutagenesis application it is recommended to have PAGE-purified primers (>40-50 nt) though at times I have used desalted oligos successfully. However, I wont recommend it since it can lead to errors at the mutagenesis site and force you to screen lot more clones in search of the correct one.
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