Single vs. Double Stranded DNA
Anton Scott Goustin
asg at cmb.biosci.wayne.edu
Sun Nov 12 16:11:16 EST 1995
juschus at novsrv1.pio1.uni-heidelberg.de (Clmens Suter-Crazzolara) wrote:
>In article <47b9b5$rhj at sam.inforamp.net>, intermed at inforamp.net says...
>>I am looking for a way to distinguish double stranded PCR products from
>>single stranded primers and the unincorporated dNTPs.
>>Is it possible to do this by measuring the 260/280 ratio?
C'mon, this is really old. Ever hear of thermal denaturation? Place SS DNA into a cuvette. Measure OD260. Remove the DNA to a tube, heat to a high temp, then return to cuvette and re-measure OD260. NO CHANGE. Try the same experiemnt with DS DNA. The OD260 will increase by ~30%. Heat treatment will not destroy the sample (if there are no nucleases around). The temperature required to get this effect depends on the length of the DS region. Less than 100 bp will denature at moderate temps (65oC for 5 min) in low ionic strength solutions. Pieces of DS DNA >1 kb may require heating up to 95oC to denature them. Warning: if the DNA contains any palindromes (invert repeats), it can "snap back", or renature as intramolecular ds DNA. This is a problem.
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