Codon Replacement - (Nov/14/2006 )
I want some opinion about codon replacement. I have a gene and its lot of rare codon. I want to replace with some frequent use codon. I want to know that its better to do through overlaping PCR reaction or through Stratagene mutagenesis kit.
I have designed Pimer for overlappiing PCR reaction but i donot have any idea to do it through mutagenesis kit. If some body have an expereicen of using Stratagen mutagenesis kit he can tell me more about it. I have to replace 11 rare codon which are widely disperse in my gene.
Yeah I have very week expression in Codon RIPL strain and rosetta strain is providing the seventh rare codon for my gene but i dont have rosetta strain and second i have some other rare codon which are not provided by rosetta strain as well. I try expression in some other promotors but there is no expression at all.
so i want to replace codon now. what you think
as this moment I have no smart ideas for such doing major repairs to a sequence. How large is your sequence anyway? Can your lab bare the cost of buying several dozen or so primers?
If rebuilding your sequence by segmental primers amplification and ligation is not possible... my suggestion would be to change expression systems.. use insect cells or yeast.
Another thing to consider is to outsource construction of the gene. For between $1 - $2 per base, there are people who will make any sequence, verify it, and ship the clone. This may not be economically feasible, but it sure beats 40 mutation reactions.