help as to why there are no bands when running my PCR product - (Nov/28/2006 )
I'm attempting to sequence a number of murine DNA samples from 2001, but am having problems with the PCR product. When I run the gel, there is only a smear and no bands. The 260:280 ratio is 2, and the primers have been used before, so the problem doesnt lie in the primers. I re-precipitated the DNA, as well as reduced the amplicon size from 502 to 340. I managed to get a band for 4 samples, yet others still show no band, and a few will show a band for GapDH, and not for my primer set. Any suggestions please?? I dont know what to try next!
Template, template, Template.... If ever there was a problem, the first suspect is always the template.
1--Is your template in good shape? Has it been degraded? Run a gel to give it a check.
2- Is your template kept in TE? (Probably is, else 1 would have occured). Thus I suggest that you dilute your template with water, start 1:20, going down to 1:100. TE is inhibits the PCR reaction. Also, the DNA is often contaminated to some degree despite great care in preparation, thus diluting the template before use, help to dilute out the contaminants. PCR amplifies the signal... it make up for the low template concentration.
3- Could add more magnesium ions. That helps too
If it is not the template, then there are more things to play with.
I agree with Perneseblue but I would add one last thing. A smear is often indicative of too much template in your PCR reaction. The way I understand it is that too much DNA will force non-specific binding of the primers resulting in a smear. Diluting your DNA 100-fold will solve the problem. Apart from that, as Perneseblue said, its possible that your template has degraded. Template is often the first port of call in these situations.
thanks for the suggestions! i'll give it a try and see what happens...