Plasmid as a PCR template - (Dec/06/2006 )
I cloned one beta-thalassemia mutation (Homo) in t-vector
but when I do ARMS-PCR ....I see 2 band...one for mutant and another
for noraml !!!!!
I have to see only one band for mutant gene!!
I change several factors...but it reapeat....
plaese help me...
It's very important!!!
umm... correct me if I am wrong here, but doesn't an ARM-PCR give you a minimum of 2 bands?
One none specific band (product of both outer primers) and at least one specific band (1 inner and 1 outer primer)
If the DNA is homozygous... so you should normally see 2 bands. If heterozygous (mixed) you see 3 bands.
Perhaps you mean, you are seeing 3 bands, 2 specific bands when only one is expected. Could the problem be with your primers? The missmatch on the -2 isn't working right. (Maybe there is more then just a single nucleotide mutation... dinucleotide mutation?). Or perhaps the nucleotide that is suppose to be bound by nucleotide at -1 of the primer isn't the nucleotide that you have predicted.
(eg... An 'A' instead of the 'T' that was predicted.)
Perhaps your plasmid is not pure? It might be a mix of both normal allele and mutant allele?
If this is a test for further ARM-PCRs of the same region, maybe you could sequence across the region, and see what is happening.
when I diluted it 1/1,000,000 in ddH2O
Its PCR result was acceptable ( only one band in mutation )
but after several days ....I saw 2 bands!!!!
I diluted it again , the similar results was reapeted!!!!
I realy don't know what I have to do ???