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-   -   Optimizing magnetic bead mix for gDNA extraction (http://seqanswers.com/forums/showthread.php?t=76871)

anjama 07-02-2017 06:59 PM

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.

Carcharodon 07-02-2017 09:56 PM

I might be stating the obvious here, but why not try 2.5M NaCl with 8% PEG? I can't explain why the other protocols seem to be working, but I really question whether it's necessary to reduce NaCl concentration so dramatically.

Also, stupid question: when you say "final mix," you mean with the sample (DNA) already included, correct?

ATϟGC 07-04-2017 06:13 AM

I have always thought of the NaCl (or another source of Na+ like Sodium acetate) as a co-factor required for an ethanol, isopropanol or PEG-base nucleic acid precipitation. NaCl might precipitate nucleic acids to a degree but I do not think that it is nearly as efficient as alcohols of PEG.

Carcharodon 07-04-2017 03:51 PM

Quote:

Originally Posted by ATϟGC (Post 208915)
I have always thought of the NaCl (or another source of Na+ like Sodium acetate) as a co-factor required for an ethanol, isopropanol or PEG-base nucleic acid precipitation. NaCl might precipitate nucleic acids to a degree but I do not think that it is nearly as efficient as alcohols of PEG.

I think that the sodium ions, in this case, may be somehow facilitating the binding of DNA to the carboxylated surface of the beads rather than playing a large role in the actual precipitation.


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