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Old 11-28-2012, 12:14 PM   #8
eladSeq
Junior Member
 
Location: Baltimore

Join Date: Dec 2011
Posts: 9
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Hi,
We are having trouble sequencing our amplicon pool due to short read failure causing very low pass filter %. For this library, I combined amplicons and then did an agarose gel extraction using a UV gel box. Then followed this with AMPure XP purification.

We have successfully sequenced a similar amplicon pool this way, so I didn't think the UV would cause a problem. The only difference between this library and the previous successful one was pooling more amplicons with different barcodes, so I guess the addition of these new primers could be causing more primer-dimers? On the fragment analyzer we only see a single clean band at 421 bp, but I guess the primer-dimers could be hiding in there by hybridizing to the full length amplicons?

Have you guys had success eliminating the short read failures by reducing the primer-dimers, or eliminating the UV gel extraction step? Any suggestions or advice appreciated. Thanks!
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