Help recovering 200 bp DNA fragment

Sergey V. Orlov Serge at bio.iem.ras.spb.ru
Fri Aug 4 17:17:06 EST 1995


     Hi, Harshinee.

A.  There is a simple and very  efficient  method  for  cleaing  and
concentrating of nucleic acids ( and other high-molecular substances).
You can concentrate your sample after elution by passing  through  dry
sephadex G10  or  G15 ( prefarebly G10 ).  The volume of sephadex must
allow your sample be soaked completely. Sephadex will swell, therefore
your column should permit this .  Wash your column with TE-buffer and
collect several  first  drops  (  1  -  3  ) containing DNA.  If it is
neÓessary, you can precipitate DNA with ethanol ( DNA precipitate more
completely from small volume ).
  I always extract samples with phenol/chloroform after elution (  this
prevent degradation  by  nucleases  and  remove  protein pollutions ).
Phenol and chloroform are removed by  sephadex  filtration  completely
and don't disturb dephosphorilation, ligation etc.

B.  Try to add in sample NaAc to 300 mM ( 0.1  V  of  3  M  solution  )
before ethanol precipitation.
 I hope, this helps.

Sincerely,  Serge Orlov
E-mail: serge at serge.stud.pu.ru



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