DNA from blood clot

RPSCOTT RSCOTT at IRRI.CGNET.COM
Tue Feb 23 20:00:44 EST 1993


From:	IN%"<@CGNET.COM:hsibul at ebc.ee>"  "hsibul" 20-FEB-1993 00:33:40.42
To:	IN%"<@CGNET.COM:methods-and-reagents at net.bio.net>"  "methods-and-reagents"
CC:	
Subj:	A Fast Method for DNA Extraction?



           Hi,netters,

   Anybody out there who has used the DNA extraction protocol published

in BioTechniques V11 No3 1991 p298 "A fast method for high-quality genomic
DNA extraction from whole human blood" ?
The authors(a group from Trieste,Italy) claim they get approx. 10 micrograms
of superb DNA from 300 microliters of blood in less than an hour.Great!
If it only would work in my hands...
          
             The method is really fast and very simple indeed:

step 1-------- take 300mml of edta-anticoagulated blood,add 600mml of 
               lysis buffer.The acting agent in the buffer is
               a cationic detergent dodecyltrimethylammonium bromide(DTAB)
               (8%DTAB in 1.5M NaCl,100mM Tris-HCl pH8.6,50mM EDTA) 
               Incubate at 68 deg C for 5 min.

step 2-------- add 900 mml of chloroform to deproteinize the aesthetically
               provocative stuff in the tube.
               Centrifuge 10,000 rpm 2 min.
               Pour the supernatant in a new eppendorf

step 3-------- add 900 mml H2O and 100 mml of 5% CTAB in 0.4M NaCl.
                        CTAB stands for another cationic detergent,
               cetyltrimethylammonium bromide(also sold by Sigma
               as hexadecyltrimethylammonium etc.)
               Now centrifuge 2 min at 10000,resuspend the expected
               pellet(which in my case is not worth of mentioning)
               in 300 mml of 1.2M NaCl to exchange the detergent
               and precipitate(the 2nd time) with 750 mml  of ethanol.
               Wash and dissolve in TE.
That's it.    
The only difference from the published protocol I was forced to
introduce was that I took 200 mml of blood,scaling all the reagents
down by 1/3 simply because we don't have 2ml eppendorfs.
The amount of DNA I succeeded to recover using the method was in
the range of 1 microgram,not 10 (or should I say 10 times 2/3).

Why should the precipitation-step be repeated?
Why not just to precipitate with ethanol after step 2?
I tried it and it gave somewhat better results.


             Any suggestions  welcome,
                                            Hiljar Sibul
                                            Estonian Biocentre
                                            University of Tartu
                                            Estonia
 
                                    
                                          
                         
                                 
                                    
                                          
                         
                                 




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