I'm trying to PCR an NES that is about~60 bp in length. The problem is that when I run it on a gel, I cannot tell if I am actually getting a product or just primer dimers. I ran a control with no template DNA to compare to but the gels looked similar with bands around the 50-60bp maker. I also tried digesting the PCR product with a site in the middle of the expected product but it doesn't change the gel much. I will try the PCR again using dilutions of the template but was wondering if there were any suggestions for telling the difference between the primer dimers and my product.

Also, if I can get my product, what are the various ways to clean it up? The PCR cleanup kit our lab has only works for products 100bp or larger.

Thanks!
Jessica

if you cannot avoid primers duplexes during PCR, insert your products in a plasmid for sequencing and therefore amplification of positive clones in bacteria

Seb_

hi

are u primers 30 bp

i think u run ur starting concentration of primers parellely. if the intensity of ur bands is higher than that of primers then u can be sure its ur product.

try using a denat PAGE with higher conc or a DGGE to single single bp differences in size

prajwal

Try running on a 4% agarose TBE gel with 10bp DNA ladder (Invitrogen). This should be more accurate.

Why not just synthesize the signal from oligos? It is dirt cheap and easy to do. It will save you the hastle of having to figure all this out.

Do a dilution series with your primers to find a conc where you still get a product but no primer bands anymore.