Protocol for Re-ethanol precipitation of already purified DNA - (Sep/27/2006 )
I have DNA that has been purified through cesium chloride preparation and have been using it for transfections and not getting good efficiency--we think there may be a problem with the DNA (maybe excess ethanol was left) and would like to take some of this already purified DNA and re-ethanol precipitate it and dry it exceptionally well to prevent excess ethanol from remainig this time...what is a good protocol to use to re-ethanol precipitate the DNA?
If you think ethanol is the problem, try checking you A260/280 ratio first before doing anything else because you will lose some DNA with every step you perform.
Put it in a vacuüm rotor for some minutes (ethanol will evaporate quicker than water, so you'll lose it).
Other than that: just perform standard precipitation of nucleic acids (discussed plenty on this forum), or do a column based cleaning of your DNA...
but whatever protocol you may use , never dry the DNA too long. Prolong drying makes DNA nearly impossible to resuspend.
I think a standard EtOH precipitation would work including 1/10th volume of NaOAc. Make sure your final EtOH volume is no less than 70%. Wash the pellet in ice cold 70% EtOH at least three times.
Leave your pellet to air dry for about 5 minutes then resuspend in 20 µL of ddH2O. Freeze on dry ice then lyophilise to dryness. Resuspend in the appropriate solution to the required concentration.