I am having difficulty developing a protocol for the successful amplification, extraction, and sequencing of ddRAD libraries from sea cucumbers (Cucumaria frondosa). Recently, I sent libraries away for QC analysis after it seemed the gel extractions (using Qiagen MinElute with a 1% agarose gel stained with GelRed) were not working, i.e. getting insufficient yields. This was despite there being visible smearing on the agarose gel. The QC analysis confirmed that there was insufficient DNA in any of the samples and adapter dimers in one sample. It was suggested the buffer may be contaminated.
To give a bit more context, these libraries were digested using Fisher Scientific Restriction Enzymes (Pst1, Mst1), ligated using Promega T4 DNA ligase, and amplified using Phusion Flash High Fidelity PCR Master Mix. The DNA extraction was performed using a Qiagen DNeasy Blood & Tissue Kit.
Has anyone faced a similar problem? Any ideas what might be going wrong? If you need more information, please ask.
Hey there Matthew,
Are you running the PCR before size-selecting (via gel excision)?
- Sean
Hi Matthew,
This issue can arise due to multiple reasons. Have you quantified your DNA extracts before setting up the digestion? Were they fine? If yes, then did you lose a lot of DNA during cleanup steps (after adapter ligation and another one after PCR)? Please do check the concentration of a few samples before and after each step (eg, ligation, cleanup, PCR, and size selection) to be sure about everything is going good.
Also, if you think you have less DNA to start with and the efficiency of size selection is not great (which is true for gel extraction), I suggest you perform size selection using Ampure beads.
Hope this is helpful.
Hi Matt,
If it's the DNA, see Tom's Tweet here https://twitter.com/Tom__Jenkins/status/1295276044228976642 looks like this salting out extraction protocol or Qiagen DNEasy PowerClean cleanup may help for RAD-seq suitable DNA prep from marine inverts?
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