PCR on degraded templates - (Aug/25/2009 )
I'm trying to amplify a 1000 bp region from human genomic DNA from a solid tumor. The reaction works very efficiently (after optimizing Mg and annealing temps) on my control DNA (human genomic DNA from Promega): I get loads of product, very clean, no non specific amplification. But when I use the same reaction conditions on my tumor sample I get no product. The tumor DNA has undergone Whole Genome Amplification (Qiagen RepliG kit), and is ~2 yrs old (stored at -20C), so it might have degraded. Unfortunately, this is the only DNA that's left from this tumor, so I really need to get this PCR to work! Any suggestions?
Take a look at the "Pre PCR" product from NEB, intended for amplification of old or poorly stored templates. I believe Epicentre also has a similar product.