Shearing of DNA

tfitzwater at gilead.com tfitzwater at gilead.com
Thu Mar 1 19:49:20 EST 2001


>Dirk Daelemans (ddaelemans at ncifcrf.gov)
>Thu 15 Feb 2001 - 21:23:27 GMT


>I would like to shear DNA to fragments of about 300-500 bp. Can anyone
>recommend an easy method that gives a nice distribution (not bigger than
>1000 not smaller than 200 bp) of sheared DNA?


>Thanks

Dissolve 1 g DNA (e.g. Salmon Testis DNA or Herring Sperm DNA, etc.) in 50
mL 0.1 N NaOH.
Add 1.5 mL concentrated HCl and mix quickly.  DNA will precipitate
immediately, and should not be stirred for more than a few seconds to
prevent formation of a large aggregate.
Incubate at room temperature for 20 minutes to partially depurinate the
DNA.
Add 2 mL 10 N NaOH (OH- concentration to 0.1 N) and stir till DNA
redissolves completely.
Incubate at 65°C for 30 minutes to hydrolyze the DNA to size range of
250-1000 nucleotides.
Cool to room temperature and add SDS to 0.5%.
Extract 2x with Tris-buffered phenol:chloroform.
Add 7 mL 3 M NaOAc pH 4.5 and mix.
Add 60 mL 2-propanol and mix.  Chill 15 minutes in dry ice/ethanol bath to
precipitate the DNA.
Spin 10K/20 minutes/4°C.  Decant and wash pellet with 10 mL 70% ethanol and
decant.  Do not dry the  DNA.
Redissolve in 50 mL 25 mM NaOH.  Store at 4°C in 25 mM NaOH to prevent
renaturation.  Do not freeze DNA in NaOH.
(T.Sargent 1988 Meth. Enz 152 p. 432.)

Tim Fitzwater
Associate Scientist
Gilead Sciences


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