We know that DNA extraction process begins with the mechanical separation of the nuclear contents from the rest of the cell, which is carried out by sonication, agitation and the addition of SDS detergents. To further break down cell components and then draw off the DNA associated proteins, researchers typically add ammonium, sodium acetate or similar salts during this stage of the procedure. Alcohol is trapping the water and facilitates Na+ to interact with the PO3-, and then precipitate DNA, so what is more efficient using of ethanol or isopropanol?
NaCl used in CTAB buffer used to remove carbohydrate.
Isopropanol or other alcohol used to precipitate nucleic acid. 70% ethanol was is used to remove residual protein and carbohydrate in nucleic acid pellet (DNA or RNA)
I found this useful protocol on the web and in general I refer always to the Molecular Cloning: A Laboratory Manual by Michael R. Green and Joseph Sambrook
Ok for the work of salt but I would like to ask what is better ethanol or isopropanol for precipitating DNA, and why?
Thanks a lot Valeria De Arcangelis for this useful protocol!
The overall function of salt and ethanol/ isopropanol is to precipitate DNA from the solution. The salts neutralize the negative charge of the negatively charged phosphate in DNA and the isopropanol /ethanol removes the hydration shell of H2O molecules around the phosphate. The total effect is that the DNA molecules can come together due to neutralization of charge and removal of water that leads to the precipitate from the solution.
Ethanol is better as compared to isopropanol for any nucleic acid precipitation. As isopropanol along with nucleic acid also precipitate salt used in nucleic acid precipitation (Na or NH4 acetate) but ethanol used to avoid the precipitation of salt at final step.
Hi Mohamed,
Please see: "DNA Precipitation: Ethanol vs. Isopropanol" [see the website for more]
(http://bitesizebio.com/2839/dna-precipitation-ethanol-vs-isopropanol/
The difference between isopropanol and ethanol is the solubility of DNA in each solvent [a sample paragraph from the site]
DNA is less soluble in isopropanol so it will fall out of solution faster and at a lower concentration, but the downside is that the salt will too. With ethanol, the DNA needs to be at a higher concentration to flocculate but the salt tends to stay soluble, even at cold temperatures.
DNA falls out of solution in 35% isopropanol and 0.5M salt. Using ethanol, the final concentration needs to be around 75% with 0.5M salt. So for the typical precipitation protocol, isopropanol is added from between 0.7-1 volumes of sample and ethanol is added at 2-2.5 volumes of sample.
I hope the website provides enough info for the question.
Alcohols generally are used for DNA precipitation but as Yuan-Yeu Yau mentioned isopropanol is stronger (harsher) than ethanol, and ethanol precipitated DNA usually of higher quality.
Salts are required for DNA precipitation alongside ethanol or isopropanol. Both alcohols are the same efficient for DNA extraction.
Regards.
I agree with Yuan-Yeu, isopropanol provides more yield with lesser concentration but with ethanol you get a higher DNA yield. I also believe choosing which alcohol somehow is related to which type of tissue you are extracting DNA from (for instance if extracting from a low quality starting material) and what is your next application after DNA extraction.
How to improve after doing extraction with isopropanol. Is it better to was the sample to reduce the amount of isopropanol?
Is anyone please tell me how to remove all salt content in magnetic beads based nucleic acid extraction. But I tried ethanol, Tris.HCl, and Guanidine as a wash buffer 1. Then Used NaCl and Tris HCl as a wash buffer 2. But have not got the purified DNA.
Dear Niranjan
Please Follow the recipe and obtained your DNA:
Wash Baffer 2: Nacl 10 mM + 10 mM Trac Base c + alcohol 70% PH adjusted 5
added to Spine + 8000 rpm2 min
remained in lab 25 degree for 5 min
then add
Elution Buffer : Buffer Tris (TE) in 60 degree adjusted PH 8
you I think you did a tiny mistake and used Elution Buffer instead wash buffer 2 and missed DNA.
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