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Old 09-10-2013, 06:54 AM   #1
Location: Edinburgh

Join Date: Jun 2013
Posts: 17
Default Extra peak after TruSeq DNA library prep

We made library preps following the standard TruSeq DNA library prep method, beginning with 3ug gDNA and took this through the steps shearing-end repair-A tailing-ligation-size selection-PCR.

We use the blue pippin for size selection and 6 cycles of amplification.

Now, we are seeing an extra peak over double the size of the intended library peak (see attached). Is this a primer-dimer peak? Or a single-stranded artefact? Do you think it will affect the sequencing of the library?

Any advice you could give me would be much appreciated.

Thank you.
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File Type: png Screen shot 2013-09-10 at 14.47.01.png (45.4 KB, 115 views)
anna_m is offline   Reply With Quote
Old 09-10-2013, 10:36 AM   #2
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Location: New England

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It's not primer dimer (that would be ~128 bp). It may just be a bubble peak from too many cycles of PCR. Sometimes, when the strands reanneal only the adapters anneal with a mismatched fragment in the middle. This creates a "bubble" and the DNA runs strangely on the BioAnalyzer.
microgirl123 is offline   Reply With Quote
Old 09-12-2013, 07:16 AM   #3
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Typically bubble products won't affect your sequencing results because the double stranded product is denatured prior to cluster generation. To verify, heat a small aliquot of the sample to 95C for 5 minutes and place on ice. The denatured product should appear as a single band on a Bioanalyzer RNA Pico 6000 Chip Kit.

- Genohub
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