Tips on ethanol precipitation of nucleic acids and wash - (Mar/13/2005 )
Hi
You should be able to precipitate DNA at room temperature with 100% EtOH, but at -80 it should have mostly precipitated within 30 minutes, although you could try leaving it overnight.
The best way to remove supernatant without distrubing the pellet is to use a 200 uL pipette (yellow tip) to remove the liquid, maknnig sure that you don't touch the pellet as you get lower in volume.
Centrifugation is probably your problem here, you will be needing to spin the DNA down at anything over 13000 G for at least 15 minutes. It also pays to have all the tubes facing the same way in the centrifuge so that you know where the pellet is so that you don't disturb it during washing and removing the supernatant.
Good luck
Bob
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.
How does glycogen works?
How does glycogen works?
It is an inert carrier. The protein is derived from the mussle. It sorta surrounds the dna and since it is a long-chain proteoglycan, easily precipitates and carries the dna is surrounds down with it during centrifugation.
It works quite well and does not interfere with downstream applications (so far as I have noticed). An added benefit it that it makes the dna pellet much easier to see. I have used it with RNA precipitation also... carried it into cDNA synthesis and pcr with no adverse effects.
Hi,
How much glycogen do you add to the reaction? What's the concentration of your glycogen solution?
Thanx,
Freiberger
The amount of glycogen needed depended on the volume of nucleic acid solution. According to Roche, 1 ul glycogen (20 ug/ul, that's 20 ug) allows to precipitate pg-amounts of DNA or RNA from a volume of 1ml.
I agree with Badcell, 70% ethanol is the right concentration for DNA and RNA, increase in the percentage won't help so much.
try this out
cheers
Hi,
How much glycogen do you add to the reaction? What's the concentration of your glycogen solution?
Thanx,
Freiberger
Hi all,
glycogen can be added at the concentration of 100ug/ml
but not needed for medium to high molecular weight DNA
I have used 65% ethanol to wash both DNA and RNA pellets. This is cutting it close (from memory 62% is the lowest you can go), but it can help if salt is a problem.
One problem using a 65% ethanol is that 100% ethanol absorbs water from the air once the stock bottle is opened. For this reason I always use 95% ethanol stock to make up my ethanol washes.
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?
Thank you.
Mario
70% will work fine, if you have DNA in your tube as soon as you put absolute alcohol and salt into it you will be able to see bubbles rising from the bottom of the tube if you tap it. this is sure way to know whether your tube has DNA. you can then precipitate it at-70 or -80 degrees for 15 min.
Then you can wash it with 1ml of 70% alcohol