I have extracted historic DNA from animal tissue using the Qiagen DNeasy kit and eluted the DNA in the provided elution buffer, which contains EDTA. I will perform whole genome sequencing on an Illumina platform. Because of the low DNA yield, I will utilize the TrueSeq Nano or Microplex prep kit. These library prep kits are sensitive to EDTA. I must therefore cleanup the extracts and use an EDTA-free elution buffer. What approach should I use for the cleanup?
I will avoid AMPure cleanup due to the small peak sizes (
Eliminating the EDTA trouble may be easier to accomplish than eliminating the EDTA. From what I understand, you have a precious enough sample (if I got right the "historic" part) which you don't want to waste. I suspect the problem can only be the chelating effect on the metals needed for enzyme activation. Thus, find out what your enzyme is, what is the co-factor metal (Mg?) and supplement using PCR grade chloride solution. Most of the times pmol traces will solve the issue.
Thank you for your suggestion! Have you tried this solution for TrueSeq Nano or Microplex prep? Considering the costs of an Illumina run, I am not sure if I am brave enough to test it..
Thank you, Albino! I am currently testing this approach with some extracts obtained from ~130-year old bird tissue. If the recovery rate is acceptable, I will try this. Otherwise, the GeneJET PCR Purification Kit might be an option.
- Badges
- Science topic
- Similar topics
- Biological Science
- Genetics
- Genetic Analysis