Help recovering 200 bp DNA fragment
Sergey V. Orlov
Serge at bio.iem.ras.spb.ru
Fri Aug 4 17:17:06 EST 1995
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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