In DNA extractions, how much is too much salt in a CTAB extraction buffer? Protocols hover around 2.5 molar; if you go over this (e.g. 25 molar), will you saturate your solution, and precipitate the DNA too early?

Background: Most DNA extraction protocols approximate the following:

  1. ~Physical lysis
  2. ~Chemical (detergent) lysis
  3. Phase separation (dreaded phenol-chloroform-isoamyl step)
  4. Precipitate, dry

Variants are plentiful, but a common feature of steps 1\2 is inclusion of CTAB and NaCl in the extraction buffer - detergents and salts help to rupture the cell membrane, but (maybe more importantly) CTAB 'selectively' complexes and precipitates out organic impurities (saccharides, proteins...) while DNA/RNA are left in solution.

For this to work, the net-negative charge of the DNA phosphate (PO4-) backbone needs to be neutralised allowing the DNA to stay in solution: sodium (Na+) ions are attracted to the regular (PO4-) groups, while more irregularly spaced charges* in proteins etc. associate tightly with CTAB, and 'fall' out of solution: you keep your DNA in the liquid phase and purify, with excess salt washed out at the end using repeated 70% EtOH washes.

Would excess Na+/Cl- effectively level the 'selective' properties of CTAB? 'Salting-Out' the DNA from solution (disrupting the solvation shell) is used in step 4, most often with sodium acetate and 2-propanol: how much salt can be used in Steps 1\2 before the DNA is precipitated out with other organic materials? Are there other considerations (e.g. salts and organic solvents in Step 3)?

First description I've found for this is Zhou (1996), but a really good protocol on it is Wilson (2001).

*: presumably?

  • $\begingroup$ What are you afraid of? Did you follow the published protocol? If not, just repeat it and follow the protocol. Ffrom the standpoint of biochemical purification, a cell contains four types of molecules: proteins, lipids, carbohydrates, and nucleic acids. These last two co-purify through most fractionation steps. As I recall, the CTAB binds to the denatured proteins. and the denatured protein-detergent complex is highly insoluble in the presence of high concentrations of monovalent cations--voila. RNA can be precipitated by monovalent cations alone, but I don't think DNA can. $\endgroup$ – mdperry Aug 21 '15 at 2:58
  • $\begingroup$ Saturated sodium chloride is approximately 6M, making 25M impossible to achieve in water. $\endgroup$ – March Ho Aug 21 '15 at 12:04
  • $\begingroup$ @ mdperry my belief was that the Na+ is (also) used to 'cloak' the negative DNA phosphate-group backbone from complexing with CTAB, keeping the DNA in a soluble 'sweet spot': my worry is that too much Na+(Cl-/AcO- etc) will totally disrupt the H-bonding between the solvent and solute, and precipitate the DNA early, at the same stage as the CTAB-protein complexes, though not necessarily for the same reason. Also think you can salt out DNA, but it needs to be cleaned up afterwards. @MarchHo - Hah, thanks! Challenge accepted. $\endgroup$ – incertae_sedis Aug 24 '15 at 12:13
  • $\begingroup$ @incertae_sedis final NaCl concentration in the extraction was increased from 0.5-2M to a dramatic 5M following advice from a colleague and greatly improved DNA yield. Not an answer as such, but suggests not enough NaCl is more likely than too much. $\endgroup$ – incertae_sedis Jul 14 '17 at 9:04

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