In article <199407071416.XAA29594 at inetnif.niftyserve.or.jp>,
HGF02633 at NIFTYSERVE.OR.JP (=?ISO-2022-JP?B?GyRAQFBETT1TPCMbKEo=?=) wrote:
> Dear Netters
> I separated the 100 bp, 50 bp, and 30 bp DNA fragments with NuSieveGTG
> low melting point agarose gel. I want to subclone the 100 bp
> fragment. I tried to purify the 100 bp fragment. However, the recovery
> is very poor (less than 30 %).
> After electrophoresis, I excised the gel band of 100 bp DNA, melt the
> gel at 65 C, did phenol extraction, and precipitated the DNA with
> ethanol (in 0.1 M NaCl). Does anybody know how to recover small DNA
> from agarose gel efficiently. Please teach me.
For recovery of a 64 bp (PCR) fragment from a 2% agarose gel I used the
protocol in TIG V7 (4) p 109 , 1991.'Fast recovery of DNA from agarose gels
by centrifugation through blotting paper'. Subsequently I precipitated the
DNA with lineair polyacrylamide, which is very efficient: NAR (1990) V18(2)
p 378. I used it also for larger fragments, and it always worked for me.
Hope this helps.
Fimme Jan van der Wal
dep. Molecular Microbiology
Vrije Universiteit Amsterdam
de Boelelaan 1087
1081 HV Amsterdam, NL
vdwal at bio.vu.nl