Tips on ethanol precipitation of nucleic acids and wash - (Mar/13/2005 )
I have a question which has been haunting my mind for a few years. About ethanol wash, almost all protocols just tell you to do a ethanol wash after precipitation but don't tell how. My questions are:
1. How much ethanol (70% for DNA, 75% for RNA) should be added?
2. Should I mix or vortex the tube after adding the ethanol?
3. Why is a lower centrifuge speed used for washing compared to the precipitation, Is higher speed OK for washing because I am always tempted to spin at high speed to be sure my DNA will adhere tighter to the wall of the tube.
4. Why 70% is used for DNA and 75% for RNA?
for your 4th question the most probalbe answer could be, RNA is more prone to dissovle inwater because of its single stranded nature and its low molecular weight.
where as double stranded DNA can with stand the disolution at 30% of water and similer is applied to RNA .
hope this answer could convince you
At which step should i add glycogen during precipitation of dna?
Add the glycogen and mix before adding the ethanol.
DNA sequencing software
ok. I have shared DNA. I have precipitated it with 100% EtOH+1/10 volume of Na Ac 5.2+1 mcl of glycogen....but it doesn't want to pellet. I can see the bubbles, I know I have DNA...what would you use? what would you increase? ETOH, salt?
I really do not understand what is happening.
I assume you mean sheared DNA. Are you getting a glycogen pellet after spinning? How much EtOH are you adding? How do you know your DNA is not pelleting anyway?
DNA sequencing protocols
Well, finally I got it. I added more glycogen(1mcg) and it made the difference.
Here are my two cents:
The precipitation should last at least for 20 min at -20C or -80C.
Before centrifugation, you should not be able to see any pellet.
To remove the supernatant, I decant the tube or aspirate the liquid using a tip connected to a vacuum.
Use glycogen as a carrier if DNA amount is very low.
i am facing a similar problem wid the extraction of plant DNA wherein i dont see any precipiatate during the addition of cold ethanol which i used to see earlier in my DNA extractions. i grind the leaves using liquid nitrogen ,wether the improper grinding affects the DNA extraction?. a point i could notice tht, whenever i get a pale green colour (might b cholorophil) in my Chloroform : isoamylalcohol extract, i dont get to see a precipitate wid cold ethanol. so do u think keeping it in
-20 degrees i will b able to see precipitate or after the centrifugation i will b able get a precipitate. if so wht speed i sholud centrifuge it (in RCF). and let me know the relationship between RCF and RPM.
I really do not understand what is happening.
I used to precipitate DNA with 2.5M Ammonium acetate(7.5M stock) and 2.5 volume of Absolute ethanol.It works well. Ammoniun acetate is easy to remove from the DNA pellet when compared to sodium acetate(which may create problem in cloning process.)
And for DNA washing, I use to wash with 300ul of 70% ice cold/ refrigerated ethanol and spin at 10,000 rpm for 15 minutes, 4 degree.
Hello, recently I have found a protocoll, which was using sodium acetate at pH 8.0 ?
Here is a short cut out:
A. T4 polymerase blunting of DNA ends
Steps 1 through 6 should be kept on ice.
1. Mix 2 µl (200 ng) WCE DNA and 53 µl ddH2O for each IP.
2. Aliquot 55 µl for each IP sample. Keep all tubes on ice.
3. Make blunting master mix on ice:
Final Conc. Stock 1x Mix
1x 10x T4 DNA polymerase buffer 11.0 µl
5 ug 10 µg/µl BSA (NEB) 0.5 ul
40 nM 10mM each dNTP 1.0 µl
1.5 U 3U/µl T4 DNA polymerase (NEB) 0.5 µl
ddH2O 42.0 µl
4. Add 55 µl of blunting master mix to all samples.
5. Incubate for 20 minutes at 12°C in thermal cycler.
6. Add 11.5 µl of 3M sodium acetate, pH 8.0 and 0.5 µl of 20 µg/µl glycogen (10 µg total).
7. Transfer reaction to pre-chilled Phaselock tubes and extract 1x with 120 µl P:C:IA (follow instructions provided by Eppendorf).
8. Transfer aqueous layer to new centrifuge tube containing 250 µl EtOH. Incubate for 30 minutes at -80°C.
9. Spin at 20,000 x g for 10 minutes at 4°C to pellet DNA. Wash pellets with 500 µl of 80% EtOH.
10. Dry pellets and resuspend each in 25 µl H2O. Chill on ice.
So my question is why did they use pH 8.0, normally it is 5.5 ? The next step would be a blund ended ligation with the sheared DNA fragments (something which gives me nightmares, but this is a differen matter). Could the pH affect the ligation reaction? Another point is that they tend to wash their precipitated DNA always with 80% EtOH. After what I have read, 70% would be better for desalting, something that could also affect proceding enzyme activities. So I don´t see the point. please enlighten me
I think there is no strict for using volume of ethanol. Usually I use 1 ml of 70% ethanol both for DNA and RNA washing. I never vortex, I just dislocate the pellet and the centrifuge for 5 min in table centrifuge (12000rpm). Sometimes I also don not centrifuge, just I wash 2 times by 70% ethanol by decanding. I think the important thing is drying the ethanol. After washing ethanol should be completely evaporate