Hi, I would like to change a 5 amino acid (max 15nt) sequence in a 11Kb plasmid. What is the maximum substitution you can successfully complete with one reaction? I have done extensive SDM before, but only for 1-3 nucleotides. Here I will be changing nucleotides throughout a 15 nucleotide sequence to change the coding sequence of 5 sequential amino acids. I was thinking of using the NEB Q5 site-directed mutagenesis kit.
Will these kits work for substitutions this large? I was going to use 20-25 nucleotides of complementary sequence downstream of the mutation for the 3' anchor.
Please let me know if you have had experience with substitutions of this size in a single reaction.