Hello all, Apologies if you receive this twice, my personal email is playing up, so I’ve sent it from my work address. I’m having afew problems with DNA extraction and was hoping someone out there may have some suggestions. As part of my PhD, I am running a qPCR looking for a papillomavirus (small double stranded DNA virus) in feline skin. I have collected just under 400 samples of plucked hairs and skin swabs from clinically normal cats, including 1 day old kittens, older kittens and adults. The samples are in 100µL 0.9%NaCl at -20⁰C (I’m only looking for DNA not RNA). I have an Ambion MagMax viral RNA Isolation kit, which despite the name, is designed to extract total nucleic acids from samples with low cellularity. The kit input volume is 50µL and the elution volume 40µL. After some practise extractions, I cannot amplify the feline reference gene (r28S DNA) from any of the samples. In contrast, when I’ve done extractions with the Roche High Pure PCR template kit (input volume 100µL and the elution volume 100µL), I can amplify the reference gene from all the samples although the copy numbers are very low (~0.05-3 copies/ µL). I have tried various other methods (Phenyl-chloroform-isoamyl alcohol extraction, NaOH, TrisKCl extractions) with some success, ie. I can amplify the reference gene from a proportion of the samples. The extracted samples have quite low DNA concentrations by spectrophotometry. Further purification or dilution of samples does not change the result. Thus I suspect the problem is that I am working at the low end of what is detectable by my qPCR rather than having an inhibitor present. My qPCR is working well, with standard curves consistently showing a r2>0.99, efficiency >90% and can detect 2 copies of the plasmid containing the reference gene. I was wondering if anyone had any suggestions as to why the magnetic bead extraction isn’t working even though in theory that should give me the best result? As I’ve already bought the kits I would prefer to get these to work, if possible, rather than buy the Roche spin columns. Thanks for your time, Neroli
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