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Old 07-02-2017, 06:59 PM   #1
anjama
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Location: Florida

Join Date: Mar 2016
Posts: 15
Default Optimizing magnetic bead mix for gDNA extraction

I've been working with the protocol in this thread for making magnetic bead mixes: http://seqanswers.com/forums/showthread.php?t=49507

I have it working well as a substitute for Ampure/SPRIselect, but I've also been experimenting with it in gDNA extraction, and it seems to work well at a 0.5x bead to sample ratio, which essentially is clearing out everything ~600-700bp and lower. But now I'm trying to tweak it so that selects at least at 1000bp using a 1.0x ratio. This paper seems to accomplish this using 0.3M NaCl and 8% Peg solution: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2572261/

So, I reduced the amount of the NaCl and Peg solutions for the protocol in my first so that the final mix is 0.3M NaCl and 8% Peg, and made up the difference with water. I ran two replicates for a variety of samples side-by-side on a single plate in a Kingfisher Flex, so they all were effectively handled identically. One replicate was the 0.5x ratio of SPRI mix, and the other was a 1.0x ratio using my new 0.3M NaCl/8% Peg mix. The first replicate worked perfectly, the second failed completely.

The 0.3M NaCl seems really low, and I thought that maybe it was too low to precipitate the DNA, so based on a document I don't have a link to at the moment, I added NaCl to my mix to bring it up to 1M NaCl. This still didn't work.

The protocol from my first link, which works, is 2.5M NaCl. Is my NaCl molarity in my new mix still too low? If so, then why are other protocols working with less? I understand that the NaCl precipitates the DNA, but I guess I don't understand what impact varying the molarity of the NaCl has on the final result; if anyone could enlighten me, I would appreciate it.
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