ERRATUM: PFGE to separate 20-50 kb

[Andy Phillips]

Chris Boyd wrote:
> 
> Andrei Popov (andrei.popov at bbsrc.ac.uk) wrote:
> : Hello everybody,
> : recently there was a posting where it was asked how to separate
> : DNA fragments in the range of 20 to 50 kb. 

...stuff deleted...

> Coincidentally, this very question is also addressed by Longo and
> Crouse in the latest Focus (Life Tech.) vol 18 no 1 pp 17-19. They
> devised a protocol _not_ requiring PFGE.  To summarise, for resolving
> fragments > 25 kb (and < 60 kb), they recommend unidirectional (i.e.,
> conventional) electrophoresis at 40 V for at least 22 h in 0.5% TAE
> agarose gels (buffer not recirculated) and stained post electrophoresis
> with ethidium bromide. I don't think the resolution is as good as
> Andrei is getting, but you can clearly distinguish 35 from 40 kb in
> their photos.
> 
> Best wishes,
> --
> Chris Boyd                       | from, | MRC Human Genetics Unit
> chrisb at hgu.mrc.ac.uk             |  not  |  Western General Hospital
> http://www.hgu.mrc.ac.uk/~chrisb |   for |   Edinburgh EH4 2XU, SCOTLAND

When I worked on chloroplast DNA, long before the invention of PFGE, I resolved 
fragments of 20-40kbp by a vary similar method to that described above: unidirectional 
elctrophoresis in 0.5-0.6% agarose gels in TAE, run at 20-30V for 36 hrs at 4oC, 
staining after electrophoresis. Worked very well. 

Andy


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