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Old 04-19-2016, 04:51 PM   #7
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Location: boulder, co

Join Date: Aug 2012
Posts: 5

we were loading less than 26pM routinely as a way to combat the affect of the aggerates, to as little as 10pM, this helped with polyclonal but had no affect on the short read fragments. The heatcool should be performed on the purified library after it has been quantified with picogreen (not bioanalyzer as the aggeration alters the size it measures and messes up your concentration calculation) and you are preparing to make the dilutions necessary to get your library to a concentration where there is 1 copy per ISP for emPCR. so heat cool right before emPCR tempalting, also the longer you let you emPCR rxn sit around after its complete (i believe ion says its good up to 1 week) the worse your data from sequencing will be, at least it was for us.
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