Help needed: genomic DNA isolation

Mark Schweder mschweder at gnv.ifas.ufl.edu
Wed Nov 18 10:57:10 EST 1992


In article <1992Nov17.180359.4039 at ucsvc.ucs.unimelb.edu.au>, u6064043 at ucsvc.ucs.unimelb.edu.au writes:
> Hi netters,
> 
> I've been trying to isolate plant genomic DNA from leaf for making a
> genomic library. The procedure I used is quite straight forward: grind
> leaf, extract, collect nuclei by centrifugation, then lyse by 2% CTAB,
> phenol/chloroform extract twice, ethanol precipitate DNA, redissolve
> in 20 mM Tris pH8, 10 mM EDTA overnight at 4C, followed by another
> phenol/chloroform/ethanol ppt. and redissolve in TE. I've been able
> to get genomic DNA >25Kb (can't tell exact size by agarose gel) without
> visible RNA contamination (I include RNase A in the extraction buffer).
> However, the biggest problem with this method is the difficulty in
> redissolving DNA after ethanol ppt. Typically, the pellete won't dissolve
> overnight or by gentle stirring of the tube. I had to centrifuge to get
> rid of the insoluble stuff and thus get very low yield (15 gram leaf
> ---> 50 ug DNA). I've also tried to enhance the solubility by heating at
> 50C for a few hours without obvious effect. Any suggestions?
> Thanks in advance
> 
> Du
> 
Here is a DNA extraction method (step-by-step for the inexperienced) that we 
have used with a number of different plants and tissues.

A) One gram of tissue is frozen with liquid nitrogen and ground to a fine 
   powder with a mortar and pestle.
B) The frozen powder is added to 15 ml extraction buffer (500 mM NaCl, 100 mM 
   Tris pH 8, 50 mM EDTA and 10 mM B-mercaptoethanol (70 ul/100 ml)) and mixed 
   thoroughly.
C) 1 ml 20% SDS is added to the solution, mixed thoroughly and incubated at 
   65oC for 10 min.
D) 5 ml 5 M KAc is added to the solution, mixed thoroughly and incubated at 
   0oC for 20 min.
E) The solution is spun at 25,000xg (14,500 RPM with SS34 centrifuge head) 
   for 20 min and the supernatant is filtered through a single layer of 
   Miracloth.
F) 10 ml 100% isopropanol is added to the solution, mixed thoroughly and 
   incubated at -20oC for 30 min.
G) The solution is spun at 20,000xg (10,500 RPM with SS34 centrifuge head) 
   for 15 min and the supernatant is discarded while the pellet is allowed to 
   dry and then dissolved in 500 ul T50E10 (50 mM Tris pH 8 and 10 mM EDTA).
H) The solution is spun in a microfuge for 10 min and the solution is 
   carefully pipetted off leaving any pellet.
I) 500 ul phenol (phenol equilibrated with 100 mM Tris pH 8, 0.1% 
   hydroxyquinoline and 0.2% B-mercaptoethanol) is added to the solution, 
   mixed thoroughly and spun in microfuge for 3 min.
J) The upper aqueous phase is carefully pipetted off leaving the aqueous/ 
   organic interface.
K) 250 ul phenol and 250 ul chloroform (24:1 (v:v) chloroform:isoamyl alcohol) 
   are added to the solution, mixed thoroughly and spun in a microfuge for 3 
   min.
L) The upper aqueous phase is carefully pipetted off leaving the aqueous/ 
   organic interface.
M) 500 ul chloroform is added to the solution, mixed thoroughly and spun in a 
   microfuge for 3 min.
N) The upper aqueous phase is carefully pipetted off leaving the aqueous/ 
   organic interface.
O) 180 ul 7.5 M ammonium acetate is added to the solution and mixed thoroughly.
P) 800 ul 100% ethanol is added to the solution, mixed thoroughly and spun in 
   microfuge for 5 min.
Q) The supernatant is discarded while the pellet is allowed to dry and then 
   dissolved in 500 ul T10E1 (10 mM Tris pH 8 and 1 mM EDTA).
R) 2.5 ul 10 mg/ml DNAse free RNAse A is added to the solution, mixed 
   thoroughly and incubated at 37oC for 10 min. Steps I - Q are then repeated.
-- 
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   Mark Schweder                | There are three things that smell like fish!
   Plant Science Laboratory     | One of them is fish...
   Agronomy Department          | The other two...
   University of Florida        | Are growing on you...
   Gainesville, Florida 32611   | (Frank Zappa, Jumbo Go Away)
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        mschweder at ifasgnv       |         mschweder at gnv.ifas.ufl.edu
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                  DISCLAIMER: I know nothing about nothing



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