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:
- ~Physical lysis
- ~Chemical (detergent) lysis
- Phase separation (dreaded phenol-chloroform-isoamyl step)
- 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)?