I'm not sure how you are doing this method exactly ... are you using primers that contain your mutation that will amplify the entire plasmid? Or are you making a PCR product containing the mutation and using that product as primers to amplify the entire plasmid?
Sometimes I have noticed that because of secondary type structure of a plasmid you actually need a longer extension time than you would think. You could try increasing the extension to see if your plasmids aren't being fully copied.
How long is your plasmid exactly, and what extension time are you using?
What do you mean by 'loads of colonies'? Hundreds? Thousands? Are you plating the entire transformation?
Are your mutagenic primers PAGE purified? That is a very important issue with this method. PCR grade primers are not good to do site directed mutagenesis.
Hi thanks for your responses. My plasmid is large, 8.9kb. I have used primers with the mutation in to amplify the whole thing.
The extension time used was 4.5 minutes (30 secs per kb) which is suggested in the Quikchange kit. I am using different reagents to this kit but the Taq I have suggests the same time. I could try longer though, I hadn't thought of that as I saw a product on my gel.
By loads of colonies I meant thousands - the know cells have a good efficiency and I just wanted to know my transformation process was working.
Thanks again!
My primers are purified, I have ordered others for a different plasmid and have got them to work fine.
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