Primer Dimers in Library

[seleenjaber]

Hi everyone, I could use some help. We're experiencing issues with our post-PCR cleanup process not effectively removing primer dimers. Currently, we're using a 1:1 bead ratio for cleanup. We tried a 0.6 bead ratio but noticed significant library loss. Do you have any tips or tricks for effectively cleaning up the library without compromising yield? Also, any advice on narrowing the library distribution would be greatly appreciated. Thank you!

[fchatonnet]

Dear seleenjaber,

a picture of an electrophoregram (BioAnalyzer analysis for example) would be nice to have an idea of your library size distribution and to be able to give better advice. I would suggest to lower the amount of primers used in your PCR reaction and then use an intermediate sample / bead ratio (providing you're writing about SPRI beads clean-up), because they are very sensitive and going from a 1:1 to 0.6:1 ratio is a huge step in size selection. Maybe trying 0.8:1 or 0.9:1 will solve your issue.
For narrowing the library size distribution, you may want to use double-size selection, while performing first a right side size selection (removing the long fragments) and then a left side size (removing the short fragments).
You can find more information, recommendations and protocols / user guide for beads here: https://www.beckman.fr/reagents/geno...elect-protocol
Good luck!