EtBr and "clean" DNA

Hiranya Roychowdhury hroychow at NMSU.EDU
Fri Jun 16 11:22:41 EST 1995


On Fri, 16 Jun 1995, Ole Skovgaard wrote:

> In article <9506121350.AA18506 at intnet.upj.com> pskaytes at INTNET.UPJ.COM writes:
> >From: pskaytes at INTNET.UPJ.COM
> >Subject: Re:  EtBr and "clean" DNA
> >Date: 12 Jun 1995 07:03:48 -0700
> 
> >  The title is
> >"A 20-Minute Ethidium Bromide/High Salt Extraction Protocol for Plasmid
> >DNA", by Willem P.C. Stemmer, in Biotechniques, about 5 years ago.
> 
> >Good luck,
> 
> >Paul
> 
> 
> If anybody find the precise ref. please post it,
> 
> thanks
> 
> ole
> 
> ******************************************
> ****   Ole Skovgaard                  ****
> ****   Dept. Life. Sc. & Chemistry    ****
> ****   Roskilde University, DK        ****
> ******************************************
> 


This is a very handy wya of cleaning up DNA and works extremely well. I 
do not have any reference with me, but here is what I do, for those who 
are interested or are having trouble with sequencing, pcr, digests etc.:

1. DNA in 200 to 250uL. 

2. Amm. acetate to 3.5M final conc. I use equal
vol of 7.M amm. acetate. 

3. Add EtBr to final conc. 0.35mg/mL (approx.)

4. Mix by inverting or very slow and gentle vortexting. Let stand at RT 
for 5 min.

5. Add equal vol of PCI and extract twice. This step removes about 80% of 
the EtBr.

6. Extract with equal vol. of CI. The remainder of the EtBr should go by 
now. If not, an extraction with TE-saturated butanol may be in order.

7. Ppt. the DNA with 2 vol. of EtOH and proceed from there as with any 
other sample.

OBVIUOS NOTE: The concentration of the EtBr renders the DNA very 
susceptible to UV damage, hence it is best to keep the tube wrapped in 
foil or to work in a dimly lit area away from the window and/or the overhead 
lab lightings.

			>>>>>>>>>>>>>>>>>>>>>>>>>>>
			  Hiranya S. Roychowdhury
   			  Plant Genetic Engineering Lab.
			  Box 3GL, NM State Univ.
			  Las Cruces, NM 88003
			  Phone: (505) 646-5785
			  hroychow at nmsu.edu
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