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Hi all,
Wondering what the current consensus is on recommended protocols for gDNA library prep from small concentrations (1-5ng) of an AT rich genome (Dictyostelium discoideum, ~23% GC). Empirically, we've had better luck with the protocol from Baym et al 2015[1] than the official Nextera XT protocol, but that only gets us to about 35% GC. Others have tweeted about reducing the extension temperature [2], or using a different (not commercially available?) transposase [3], but I thought I'd ask the hive mind prior to setting off on my own experiments.
I'd love to be able to do a PCR-free protocol, but the experiment I'm planning doesn't give more than about 5ng of gDNA, so that's off the table for all the kits I've seen.
Thanks!
[1] https://journals.plos.org/plosone/ar...l.pone.0128036
[2] https://twitter.com/masonry_stoves/s...29693978722304
[3] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5240201/
Wondering what the current consensus is on recommended protocols for gDNA library prep from small concentrations (1-5ng) of an AT rich genome (Dictyostelium discoideum, ~23% GC). Empirically, we've had better luck with the protocol from Baym et al 2015[1] than the official Nextera XT protocol, but that only gets us to about 35% GC. Others have tweeted about reducing the extension temperature [2], or using a different (not commercially available?) transposase [3], but I thought I'd ask the hive mind prior to setting off on my own experiments.
I'd love to be able to do a PCR-free protocol, but the experiment I'm planning doesn't give more than about 5ng of gDNA, so that's off the table for all the kits I've seen.
Thanks!
[1] https://journals.plos.org/plosone/ar...l.pone.0128036
[2] https://twitter.com/masonry_stoves/s...29693978722304
[3] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5240201/