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
>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
Probably again depends on the G/C and secondary structure content, a bit
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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