Site directed mutagenesis - Site directed mutagenesis (Oct/07/2009 )

Hi,

I have been trying cloning to obtain a construct for the past 7 months and it has not been working successful. Since I'm digesting the backbone as well as the plasmid that I obtain the fragment (insert) with the same enzymes I have been getting constructs with the flipped orientation but not what I want.

My question is: Does anyone know performing site directed mutagenesis on a plasmid ~12Kb is possible. Since I have heard that using a bigger plasmid would require more PCR cycles which would potentially introduce more mutations. Earlier the plasmid that I made the mutations was about 7Kb and it only had the specific mutation that I was interested, and i digested out the fragment that I wanted including the mutation and was trying to clone it to the 12Kb Plasmid which is pretty much identical to the 7Kb with a few nutrient markers, and is a expression vector which is where I need to finally have my mutation in.; and I was thinking of making the mutation in this 12kB plasmid itself but I was previously advised not to due to the inco-operation of unwanted mutaions and since it will be difficult for me to sequnce the entire plasmid due to the lab of a proper sequence which would lead to problems in finding primers. But I still want to try this method. Any feed back is appreciated.

If your lab can afford it, try Stratagene's Quickchange XL kit- is about $300. You can mke all the site directed mutants you want- The kit is idiot-proof and I have successfully designed elaborate site directed mutants in plasmids as large as 20KB.