PCR ghost bands - (Jun/06/2009 )

Hi all,

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.

Any suggestions...?

Hi,

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.

AC