Hello, I am working with worm genotyping recently and experienced some troubles which made me very frustrated >:(
I had been using a pair of primer for genotyping my crossed worms, usually I could always get a very nice band. But for unknown reasons, that primers no longer work well, it either gave me very weak bands or no bands. I am pretty sure I did not change anything. ( I used the same PCR machine, same program , same protocol for breaking the worms)
Anybody has the same problem before or can gave me some instructions?
Thanks in advance!
if all the other components work (Proteinase K, Taq, buffers, etc) the 2 most likely causes might be that your primers have degraded (can happen after several cycles of thawing freezing primers), or you have primer dimers (which enrich if you run 100s of PCRs with the same fragment).
I recommend you construct new primers, outside of your current fragment.
(To avoid above problems, aliquot your primers in several tubes and separate mixing/running & opening PCR-tubes/loading/running gels in different rooms.
Next time , keep an aliqout of your PCR -work-well products in stock for your primer check positive control.
or now just use genomic DNA to check your primers. Primers could be degraded, but check them firstly.
use new PCR master mixture , check first by genomic DNA with primers.
3). to see if your worm was put really into the lysis buffer under the dissect microscope.
good luck with your exp.