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Old 01-29-2018, 08:03 PM   #3
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,232
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Quote:
Originally Posted by kgoglin View Post
We have a theory that large libraries, specifically those with fragments trailing off to >2kb, do not quantitate accurately from Bioanalzyer. We think this because qPCR concentrations do not correlate. Has anyone else had this problem? It seems if we are able to remove the "tail", then qPCR matches a bit better with Bioanalzyer.
This depend on qPCR cycle settings. Using standard protocols gives enough time for extension of around up to 900 bp fragments so larger fragments are not amplified by qPCR. Since fragments above this size do not cluster efficiently they can be ignored. For accurate quantification average fragment length from the shortest fragment to 900-950 bp on the bioanalyser can be used to calculate concentration.
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