PCR ghost bands - (Jun/05/2009 )
I am frustrated because I am trying to identify the sex of e18 rat fetuses by clipping their tails and running a PCR reaction with primers designed by Primer-BLAST (NCBI) in the Y chromosome gene TSPY1.
My problem is, the gel I ran for my first PCR shows that my 13 of my 14 embryos were male - a result that, while possible, I don't believe in because
1. I can't be that "lucky"
2. some of the bands are very faint.
When I reran the PCR, I got bands for all 14 samples. These bands are all the same size but they do vary in intensity.
I prepare a master mix for the PCR and I add my template DNA after distribution of the PCR buffer.
I know it's not contamination of my PCR reagents because I have a negative control (genomic DNA isolated from a female rat) that does not show a band.
The only possibility is that I contaminated my templates while extracting my genomic DNA, but I have always made sure to handle my "suspected females" before my males.
Are you sure that you are using the correct Taq polymerase? Because for me I got an extra band on my stuff when using Qiagen HotStart Plus Taq but no extra band when I use Qiagen HotStart. So you may try a different Taq to see what will happen.