2 replies to this topic

[st-pete]

Hello,
what are the possible undesired fragments that you can get when you are doing a mutagenesis PCR and what are their origin? I have noticed that when I try introducing a mutation in a gene my mutagenesis PCR sometimes amplifies the gene without the mutation, is this due to the long product of my first PCR being amplified exponentially too?

[Rsm]

Hi,
I guess you are using your gene in a vector as template for the mutagenic PCR? Do you do do direct site-directed mutagenesis or something like overlap extension PCR? Did you remove your old plasmid with DpnI?
Cheers,
Minna

[st-pete]

Minna, on Sep 2 2009, 04:44 AM, said:

Hi,
I guess you are using your gene in a vector as template for the mutagenic PCR? Do you do do direct site-directed mutagenesis or something like overlap extension PCR? Did you remove your old plasmid with DpnI?
Cheers,
Minna

I use four PCR primers (5', C53S-3' and 3', C53S-5') to amplify two fragments of my gene (from a vector containing the gene): the 5' fragment with the C53S (cysteine53 to serine) mutation at its 3' end and the 3' fragment with the C53S mutation at its 5' end. I purify my fragments (by cutting them out of an agarose gel and purifying them with a kit). I then do a PCR with the 5' and 3' primers to amplify the whole gene with the C53S mutation.

5' primer               C53S-5' primer
   -->                             -->
5'----------------------------X-------------------------3'
3'----------------------------X-------------------------5'
                                    <--                        <--
                            C53S-3' primer           3' primer

When I insert the gene in a vector and transform it in E.coli, I, however, get a bit of the wild-type gene (the gene without the C53S mutation), how could this be possible? Could it be the long product of my first PCR (one of the early products produced in the first cycles of the PCR) that is amplified  exponentially in my second PCR?