Fw: Fw: Fw: What is the effect of a 5' overhang on gel mobility?

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Mon Jan 15 08:29:03 EST 2001


In article <200101121759.f0CHxIc13616 at ns.qnis.net>, the eminent Deanne
Bell at BIOSCI/MRC Human Genome Mapping Project Resource Centre wrote
>Actually - this seems like a good opportunity to ask a question about
>molecular markers.
>
>I am always a little sceptical as to whether the fragments in a ladder are
>really what they claim.  How often does a digestion really create fragments
>exactly 400bp, 500bp, 700bp and 1000bp in length?

I would hazard a pretty good guess that, with in vitro mutagenesis being
relatively straightforward nowadays, one could position RE sites exactly
at the right place to generate the correct sized fragments.

>
>If your lab switches over to using another ladder  - is there really
>continuity in your size calling? 

Should be but who knows what secondary structure is in either marker
such that there may be fragments running at slightly the wrong size. 

>
>If you are using a ladder say from 200bp - 2500bp on agarose gels, how will
>that ladder perform on non-denaturing acrylamide gels?  AND on denaturing
>acrylamide?

Probably again depends on the G/C and secondary structure content, a bit
like sequencing.

Duncan
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
GeneSys Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk






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