I am looking to make a single mutation but the site I want to use has the sequence aaggactgg
- PAM tgg
- mutation agg >GAA or GAG
If I use the tgg PAM then the codon can't be changed in the repair template because it is a TRP
Or Should I use the agg PAM site?
Hi Trevor,
There are a few ways to introduce a silent mutation. The easiest may be to remove a small fragment that surrounds the agg with restriction enzymes and replace with a designed DNA fragment or linker fragment made from two complementary DNA fragments (If you want to do this I can send you a protocol). Do you have this sequence in a plasmid, if so, do you know if there are unique restriction sites that surround the agg? If you post more of the sequence that surrounds the agg and any unique surrounding restriction sites I would be happy to help you out.