Comments on: The Basics: How Ethanol Precipitation of DNA and RNA Works http://bitesizebio.com/2007/12/04/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/ Daily tech tips, news and comment for molecular and cell biologists Thu, 09 Sep 2010 18:03:33 +0000 hourly 1 http://wordpress.org/?v=3.0-beta2-14565 By: Marie Bonnet http://bitesizebio.com/2007/12/04/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/#comment-19984 Marie Bonnet Wed, 11 Aug 2010 11:53:45 +0000 http://bitesizebio.com/2007/12/04/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/#comment-19984 Hello, Thanks a lot for the great post and discussions! I would like some advice to purify very small DNA fragments. In my tube I have the 40 bp DNA I want to purify from a mix containing dNTPs, proteins, salts and 10-bases oligonucleotides. and I don't want to use a gel purification step as I will do this on a large scale. I plan to increase the time of incubation on ice and the time of centrifugation as recommended by Nick. my questions are: - if I also add MgCl2, can it interfere with further enzyme restriction cut? - is there a better salt to use for small DNA? sodium acetate or ammonium acetate? - are there any other tips to increase the yield or the purity of the sample? thanks a lot for your help! Marie. Hello,

Thanks a lot for the great post and discussions!

I would like some advice to purify very small DNA fragments.
In my tube I have the 40 bp DNA I want to purify from a mix containing dNTPs, proteins, salts and 10-bases oligonucleotides.
and I don’t want to use a gel purification step as I will do this on a large scale.
I plan to increase the time of incubation on ice and the time of centrifugation as recommended by Nick.
my questions are:
- if I also add MgCl2, can it interfere with further enzyme restriction cut?
- is there a better salt to use for small DNA? sodium acetate or ammonium acetate?
- are there any other tips to increase the yield or the purity of the sample?

thanks a lot for your help!

Marie.

]]> By: How Silica Spin Column DNA and RNA Preps Work | Bitesize Bio http://bitesizebio.com/2007/12/04/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/#comment-19751 How Silica Spin Column DNA and RNA Preps Work | Bitesize Bio Mon, 28 Jun 2010 16:07:19 +0000 http://bitesizebio.com/2007/12/04/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/#comment-19751 [...] the nucleic acids you are searching for. If you used an SDS-containing detergent in lysis, try using NaCl as a precipitant to avoid contamination of the DNA or RNA with [...] [...] the nucleic acids you are searching for. If you used an SDS-containing detergent in lysis, try using NaCl as a precipitant to avoid contamination of the DNA or RNA with [...]

]]> By: Aye Riaz http://bitesizebio.com/2007/12/04/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/#comment-19711 Aye Riaz Tue, 22 Jun 2010 06:08:27 +0000 http://bitesizebio.com/2007/12/04/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/#comment-19711 Hi This is a really great site. Fell in love with it instantly. Have a question: What effect does the grade of ethanol being used has on the precipitation? For example, reagent grade vs. crude ethanol. I've had good DNA recovery usin crude etoh in our lab but would it affect downstream applications like restriction digestion? Hi

This is a really great site. Fell in love with it instantly.

Have a question: What effect does the grade of ethanol being used has on the precipitation? For example, reagent grade vs. crude ethanol. I’ve had good DNA recovery usin crude etoh in our lab but would it affect downstream applications like restriction digestion?

]]> By: Nick Oswald http://bitesizebio.com/2007/12/04/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/#comment-19540 Nick Oswald Tue, 01 Jun 2010 08:25:36 +0000 http://bitesizebio.com/2007/12/04/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/#comment-19540 Hi Natasha, Yes, I normally make up a solution and store small aliquots at -20 Hi Natasha,

Yes, I normally make up a solution and store small aliquots at -20

]]> By: Natasha Kumar http://bitesizebio.com/2007/12/04/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/#comment-19537 Natasha Kumar Mon, 31 May 2010 22:58:43 +0000 http://bitesizebio.com/2007/12/04/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/#comment-19537 Hi Nick, great article. Can you make up sodium acetate and store at 4C or -20C for long periods?? Hi Nick,
great article.
Can you make up sodium acetate and store at 4C or -20C for long periods??

]]> By: Jonathan Perreault http://bitesizebio.com/2007/12/04/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/#comment-19501 Jonathan Perreault Thu, 27 May 2010 18:23:15 +0000 http://bitesizebio.com/2007/12/04/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/#comment-19501 concerning LiCl precipitation, Ambion has a nice webpage: http://www.ambion.com/techlib/tb/tb_160.html it looks like it doesn't really inhibit translation (also, you apparently don't need to add ethanol, just 2.5M LiCl is enough) concerning LiCl precipitation, Ambion has a nice webpage:
http://www.ambion.com/techlib/tb/tb_160.html

it looks like it doesn’t really inhibit translation (also, you apparently don’t need to add ethanol, just 2.5M LiCl is enough)

]]> By: BA FR http://bitesizebio.com/2007/12/04/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/#comment-19489 BA FR Wed, 26 May 2010 08:14:01 +0000 http://bitesizebio.com/2007/12/04/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/#comment-19489 What kind of desalting or precipitation do you suggest? What kind of desalting or precipitation do you suggest?

]]> By: BA FR http://bitesizebio.com/2007/12/04/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/#comment-19488 BA FR Wed, 26 May 2010 08:13:19 +0000 http://bitesizebio.com/2007/12/04/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/#comment-19488 Hey! Great article, great discussion! Simple Question: i run a genemole RNA-Extraction and i got my totalRNA sample in TRIS (100µl). I need it desalted for RNA-Chip analysis but be especially interested in microRNAs, so i fear any loss! Hey! Great article, great discussion!

Simple Question: i run a genemole RNA-Extraction and i got my totalRNA sample in TRIS (100µl). I need it desalted for RNA-Chip analysis but be especially interested in microRNAs, so i fear any loss!

]]> By: Kenneth Mitton http://bitesizebio.com/2007/12/04/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/#comment-19428 Kenneth Mitton Sat, 22 May 2010 19:40:11 +0000 http://bitesizebio.com/2007/12/04/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/#comment-19428 We have added glycogen after the isopropanol (1 vol) (or 2 vol of ETOH), and it will increase your yield. What small amount of DNA you have that will resist precipitation will have to share that soluble compartment with the glycogen. You can add it at 37 C to your samples, mix, and then cool them down again. You also benefit from the post precip wash steps because the larger more intact pellet you get will prevent those thinner DNA residues from fragmenting and becoming physically washed away during the 70% ETOH washing. People think the yield is all about the precipitation step, but I am constantly showing staff and students how I can recover twice the DNA they recover from the same solutions simply by taking much more care of the pellet (or residue) once you get it. I make my staff "soak" (wash) three times, not twice, and not try to take off every uL of wash. The last bit dries away fast in the spin vac. We have added glycogen after the isopropanol (1 vol) (or 2 vol of ETOH), and it will increase your yield. What small amount of DNA you have that will resist precipitation will have to share that soluble compartment with the glycogen. You can add it at 37 C to your samples, mix, and then cool them down again.

You also benefit from the post precip wash steps because the larger more intact pellet you get will prevent those thinner DNA residues from fragmenting and becoming physically washed away during the 70% ETOH washing.

People think the yield is all about the precipitation step, but I am constantly showing staff and students how I can recover twice the DNA they recover from the same solutions simply by taking much more care of the pellet (or residue) once you get it. I make my staff “soak” (wash) three times, not twice, and not try to take off every uL of wash. The last bit dries away fast in the spin vac.

]]> By: Roberto Rosati http://bitesizebio.com/2007/12/04/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/#comment-19404 Roberto Rosati Sat, 22 May 2010 01:57:37 +0000 http://bitesizebio.com/2007/12/04/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/#comment-19404 Trying doesn't hurt. In your case though, I don't know if it's the right thing to do. The background idea is that glycogen will start nucleation (i.e. formation of many tiny precipitate particles) and then the DNA will stick there, helping to grow the particle and precipitating along. So if the glycogen isn't well dispersed, it'll form few nuclei, and the increase in yield won't be as much. You'd need to make sure that the glycogen forms very small precipitate particles, say, by vortexing very well - but if you're extracting gDNA... that's against your interests, I guess? If I might suggest an alternative, linear acrylamide works as well as glycogen and is cheap, quick and trivial to make. I use it with good results. I follow this protocol: http://www.ncbi.nlm.nih.gov/pubmed/2326177 Trying doesn’t hurt. In your case though, I don’t know if it’s the right thing to do. The background idea is that glycogen will start nucleation (i.e. formation of many tiny precipitate particles) and then the DNA will stick there, helping to grow the particle and precipitating along. So if the glycogen isn’t well dispersed, it’ll form few nuclei, and the increase in yield won’t be as much. You’d need to make sure that the glycogen forms very small precipitate particles, say, by vortexing very well – but if you’re extracting gDNA… that’s against your interests, I guess?

If I might suggest an alternative, linear acrylamide works as well as glycogen and is cheap, quick and trivial to make. I use it with good results. I follow this protocol: http://www.ncbi.nlm.nih.gov/pubmed/2326177

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