Doublet after real time PCR! - (Apr/29/2005 )

HI,

I'm getting a doublet (one band the size i want & another larger fragment) after real time PCR with TaqMan probes on the smartcycler. the primer set work perfect with standard PCR. Has anyone any ideas? Is it a problem with the probe? Could i try and do a standard PCR with primers & probe just to see if i get this second band?
Thanks

Dear amf,

What is the MgCl2 concentration that you used in both standard PCR and real-time PCR?

What is the size of both bands?

Regards
Yong

Hi Yong,

The band sizes I am getting are 184bp (the correct size) and ~600bp.

MgCl2 conc for standard PCR is 1.5mM
The problem with real time is that I'm using the Rooche TaqMan kit (so I don't know the final MgCl2 conc).

I went back and did a standard PCR with my primers & probe as well in the mix-it was perfect. Does this mean that my probe is ok & that optimisation is needed on real time??? My Ta was 55C, which is quite low-i'm thinking of increasing it to 60C