No PCR product after BSP, but dimers - (Sep/02/2005 )
I have been attempting the same BSP for about three months, and with no positive results. I have tried about 20 different primer pairs (12 of which were working, published sequences), and have had no luck. Previously I was getting primer dimers, then no product at all, and now I am getting primer dimers using a primer set for unmodified DNA, but nothing for the reactions using bis-modified DNA. I have tried nested PCR, HotStart with Amplitaq gold, varying primer concentrations, template concentrations, digest vs.shearing before bis-treatment, and nothing seems to be working. I had originally thought that my problem might lie in the bisulfite treatment, but the only difference between my protocol and those posted on this site is a 5hr incubation with bisulfite, as opposed to 16 (and this time has been tried and true by previous lab members). Any suggestions as to why I'm seeing these dimers/blank lanes would be appreicated.
there may be no template there to start with,
what starting amount of gDNA are you using for your modification reactions?
2ug is a good starting point.......there isn't a way of knowing before the pcr reaction if you have some template there.
Have you tried performing a magnesium titration? as magnesium conc can affect the nature of the reaction.
a titration between 0-3mM final concentration in increments of 0.5mM should somewhere within this range, yield a band.
Another method could to be to set your Tm in your PCR much much below the published, calculated Tm. That has worked in some cases for me in the past.
Try lowering your extension temperature to 64C and extending your extension time a bit, especially if you are working with a low-GC organism.
This seems very similar to the problem I have. sorry, have no idea what to do...
I actually got my PCR to work, so maybe what I did can help you. I used a temperature gradient, and found that the optimal temperature was 7 degrees below the annealing temp that had been published. So gradients are one helpful tool. Also, this PCR was nested- even though the previous researcher had gotten product after the first round, I did not, and I think a nested approach made a big difference. Also, I used a MgCl2 titration, and only added 1ul of 10mM dNTPs per 50 ul reaction. These are small things, but may make a difference. Good luck- I know how frustrating this is.