Purify large fragment from Agarose gel >10 Kb

Hiranya Roychowdhury hroychow at NMSU.EDU
Tue Oct 21 10:14:10 EST 1997


At 04:46 PM 10/20/97 -0700, Ke-Xue Huang wrote:
>Hi, Netters,
>
>Does anybody out there any give me some references or any methods, kits, 
>which work well for purification large fragment from agarose gel?(>10 kB)
>
>We met unstable results by using Geneclean or Qiagen kit.
>
>Thanks!
>
>
>Huang


For 10kb+ plasmids one has to be very gentle. I have used glass-fine
purification (as in Geneclean) on such large plasmids. The trick is to wash
the glass-fines (glassmilk) simply by diffusion over a long period of time
(typically o/n with two or three changes of the New Wash. Also, recovery is
enhanced by longer (20 min) elution at 65 C in TE or 0.5xTE.

Alternatively, you can use DEAE membrane to "trap" the plasmid directly from
the gel. S&S sells NA-45 membrane. The procedure is also known to someas
"tombstoning". This is probably the best method for isolating any size
plasmid below 15kb as it yields very clean and intact DNA.

The other method that comes to mind is the centrifugal extrusion of the
plasmid directly from the gel slice. If you need details of this protocol
let me know (or you can find it in the FAQ list I think). This is the
cheapest way to get large fragments very quickly (less than 30 min,
including clean up). I used to use this method regularly to obtain undamaged
lambda DNA.

Have not used any kit for plasmid purification (I think it is waste of
money), so can not comment on that.


Dr. Hiranya Sankar Roychowdhury
Plant Genetic Engineering Lab.
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu




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