I've heard from some people that SPRI is not a great way to concentrate DNA products. I usually use 0.6x SPRI on 40-50 uL of PCR product and elute into 30-40uL of water. I am wondering if I could concentrate the product further by just using lower volume of water (or TE, or any elution buffer), like maybe 10uL. Anyone here has any experience doing that? A lot of protocols seem to suggest putting the elutted product on a column to concentrate, which I think kinda defeats the purpose of using SPRI...
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I usually take a 50 ul PCR reaction and elute into 22.5 ul. Ten ul seems extreme - you may have problems pipetting up the liquid without beads. -
I elute 50 uL PCR products in 20 uL pretty regularly without any issues or loss in yield. When going below 20 uL, I start to see a drop in total recovery.Josh KinmanComment
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CRISPR/Cas9 sparked the gene editing revolution for both research and therapeutics.1 But this system still showed severe issues that limited its applications. The most prominent were the heavy reliance on PAM sequences, delivery limitations, double-stranded breaks that prompt unintended edits and cell death, and editing inefficiency (both in targeting and in knock-in reliability).
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