one nucleotide in the primer is wrong - (Jun/03/2007 )
I have bought one pair of primer to detect the NS5B gene of CSFV. In the process of giving the sequence, I made a mistake of giving a wrong nucleotide ( just one nucleotide of Reverse primer).
Then, I use them for my RT-PCR with the Possivetive control and my sample. The result show that:
They are specific for the CSFV in the Possivetive control
But with my sample, there is not the specific band as I need. there are 2 unspecific bands with lower weight.( I guested that is the products of DNA amplification).
So anyone can explain to me the problem was come from the primer or not.
Thanks in advance!
Then, I use them for my RT-PCR with the Possivetive control and my sample. The result show that:
They are specific for the CSFV in the Possivetive control
But with my sample, there is not the specific band as I need. there are 2 unspecific bands with lower weight.( I guested that is the products of DNA amplification).
So anyone can explain to me the problem was come from the primer or not.
Thanks in advance!
I am afraid you have to change your primer and repeat your PCR. I think your bands are not specific for NS5B gene and you have amplification for another gene that have the same sequence of your primer.
To be in the safe side change your primers because even if they gave you the results that you need, you will not be sure of your work and if you were a postgraduate student the supervisor might let you repeat your work
You might not have noticable problem if the error is at the 5' end of your primer, but if it's in the middle or at the 3' end, there'll be a problem. Anyway, re-order the primer as suggested by HAA.