I am having trouble with Genotyping.
I have been doing the same genotyping that been working well for two straight months.
However, after changing to a new tube of PCR buffer (call it A), the PCR stopped working.
I can only get smears now. (no primer band at the bottom.)
Originally, i thought buffer(A) was the cause,
since changing the PCR buffer to an older (B) gave me at least nonspecific bands with faint primer band.
However, I can use buffer(A) for other genotypes and it works as well as it did before.
And even with (B), for this one genotype, the wt/mut band does not show and it's with a smear.
So, I thought it may be a combination of the buffer and primer. (I have been using the same aliquot for awhile.)
But, even after I made fresh primers, I saw no primer-, wt/mut-, or nonspecific band. Just got rid of the smearing.
Even with buffer (B), I didn't get any mut/wt band.
Since the PCR conditions and Primer concentration/amount has been working well in the past, I don't think the conditions are all of the sudden a problem. So, I have ordered a new PCR buffer, but I am not sure what the exact cause is and wondering if anyone had an insight in this.