Hello Tamjeed,

when using the "QuikChange II XL Site-Directed Mutagenesis Kit" from Agilent, for example, you should use 125 ng (equals 15 pmole if the primer is 25 bp long) of each oligonucleotide per 50 microliters of total reaction volume. When using this kit I usually sequenced three independent colonies (at least two out of three were usually positive).

Best regards

Hello Tamjeed,

we are using the Q5 site-directed-mutagenesis kit and we use non-overlapping primers. I guess your 2-3aa are in close proximity, that would mean, that the introduced mutations are at the 5' end of your primers. The kit recommends 500µM for each primer and 1-25ng template DNA (I tend to take 1mM each primer and 25ng template).

I usually screen 5 colonies of which 1-4 are positive (depending on the complexity of the introduced mutations and -I guess- GC content of the sequence)!

good luck,

Christine

• Hi Christian, Your recommendation worked splendidly. Thank you very much.

Christine: Actually I was trying introduce mutations on two different sites that are quite far apart and require 2 sets of primers.