Amplicon resolving differently between bioanalyzer and tapestation

Pab77 · 01-25-2016, 12:08 PM

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!

microgirl123 · 01-25-2016, 12:56 PM

I have run a lot of amplicon samples on the Bioanalyzer and never seen that! Since it's a high sensitivity chip, is it possible that the previous run contaminated the electrodes with something 370 bp in length? I'd pull out the electrode cartridge, clean it thoroughly, and try again.

Pab77 · 01-25-2016, 01:18 PM

Thanks for the reply! We actually ran two other samples in this same chip and those were not contaminated with this same peak. It's unlikely that only this pin was contaminated. We can try to redo the run but yes, it's really weird.

kerplunk412 · 01-25-2016, 03:41 PM

I say re-run it and see what happens. With something this strange I always like to make sure the result is repeatable before looking too much into it.

Also, if you still have the rest of the 1:20 dilution run it on an agarose gel and see if the banding pattern repeats. There is a chance something strange happened during the dilution.

Edit: One more thing. Was anything of this size (or expected size) run on the same chip? Maybe the well got double-loaded.

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01-25-2016, 12:56 PM	#2
microgirl123 Senior Member Location: New England Join Date: Jun 2012 Posts: 200	I have run a lot of amplicon samples on the Bioanalyzer and never seen that! Since it's a high sensitivity chip, is it possible that the previous run contaminated the electrodes with something 370 bp in length? I'd pull out the electrode cartridge, clean it thoroughly, and try again.

01-25-2016, 01:18 PM	#3
Pab77 Junior Member Location: Seattle Join Date: Feb 2012 Posts: 9	Thanks for the reply! We actually ran two other samples in this same chip and those were not contaminated with this same peak. It's unlikely that only this pin was contaminated. We can try to redo the run but yes, it's really weird.

01-25-2016, 03:41 PM	#4
kerplunk412 Senior Member Location: Bioo Scientific, Austin, TX, USA Join Date: Jun 2012 Posts: 119	I say re-run it and see what happens. With something this strange I always like to make sure the result is repeatable before looking too much into it. Also, if you still have the rest of the 1:20 dilution run it on an agarose gel and see if the banding pattern repeats. There is a chance something strange happened during the dilution. Edit: One more thing. Was anything of this size (or expected size) run on the same chip? Maybe the well got double-loaded.