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Old 01-25-2016, 11:08 AM   #1
Pab77
Junior Member
 
Location: Seattle

Join Date: Feb 2012
Posts: 8
Default Amplicon resolving differently between bioanalyzer and tapestation

Hello,
We are seeing a discrepancy between the Bioanalyzer and Tapestation for a large amplicon product that we received for sequencing. We received the PCR reaction for this amplicon and did an Ampure XP bead clean up. The PCR product was run on an agarose gel before giving us and a single band of the expected size (~665bp) and primer dimers (~100bp) was seen. When we ran in the bioanalyzer (attached ppt), there are two peaks showing up. One around 370bp and the other 664bp. On the tapestation (attached ppt) we only see a single peak around 670bp. What's causing this difference? Is the amplicon making secondary structure and migrating slower in the bioanalyzer? But in tapestation, why isn't the same thing showing up? Have anyone else seen this before?
Just wanted to mention that the primers were ~30bp long and didn't have any ssDNA structures like the Y adapters in Illumina. This is a cDNA amplicon product that we are planning to ligate adapters to.
Thanks in advance!

Last edited by Pab77; 01-25-2016 at 11:13 AM.
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