I want to introduce 2 mutations using Agilent's multi-site mutagenesis kit, I designed the primers using their online tool and it gave me 2 sets for primers (1 set for each mutation) so I was wondering if I have to do this is 2 steps? i.e introduce 1 mutation using first set of primers and then then use that as a template for introducing the 2nd mutation with the 2nd set of primers or can I add all 4 primers together in a single PCR step? My understanding is that with the multi-site kit I should be able to do this in one step but confused as to why they gave to2 sets of primers? Attached is a screenshot for reference.
If I understand the form, it looks like you have submitted a request for primers that would delete all residues between 86D and 90D.
If you want to change one amino acid to another, you need to input which residue you want to change AND what you want to change it to (e.g. do you want to change 86D to an alanine?).
Good luck!
Hi Katie, Yes I did submit the primer design for substitutions D86A & D90A (the screenshot that I took was after I made the selection so it doesn't show :))....and thats when it gave me 2 sets of primers, so I was wondering if I need to do the PCR step sequentially (to get one AA mutation, and then use that as a template to get the other) or add all for primers in a single PCR reaction to get both mutations simultaneously in the final product.
To clarify, since I am planning to use the multi-site kit, I was expecting to get 1 set of primers that include both substitutions and not 2 sets of primers...
My best guess is that it was not possible to create 1 set of primers that you make all of the changes (too far apart? annealing temp too low?). You'll need to do a sequential mutagenesis (change 1 locus, then the other).
I bet their website will give an exact protocol for how to do a sequential mutagenesis. Try it both ways (change 86 then 90; change 90 then 86) and see which one works first.
Good luck!
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