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

Deanne Bell dbell at qnis.net
Fri Jan 12 12:57:14 EST 2001


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?

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

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?

Thanks for the input
TTFN
D. Bell


----------
> From: Dr. Duncan Clark <Duncan at nospam.demon.co.uk>
> Quite simply, when you run restriction fragments on gels they are never
> usually denatured! Denaturing gels are primarily run for sequencing.
> 
> Restriction fragment gels are usually agarose based and sequencing gels
> are always acrylamide based. Small restriction fragments are run on non-
> denaturing acrylamide gels.
> 
> Agarose gels will not resolve 500bp fragments to better than say 10bp
> and above that you are talking about maybe resolution of 100bp for 10kb
> fragments.
> 
> 4bp will only resolve on very small fragments on high percentage agarose
> or on acrylamide.
> 
> But will a 4bp overhang matter?
> 
> I'm guessing it will matter only if you have small fragment run on
> acrylamide gel when the resolution of the gel is high enough to show it.
> But even then I'm not sure whether a ds restriction digested DNA
> fragment with a 4bp 5' or 3' overhang will actually run different from
> the same fragment but blunt i.e.
> 
> will
> 
> ***------------------------------- 
>    -------------------------------***
> 
> run at the same size as an identical but blunt sequence
> 
> ----------------------------------
> ----------------------------------
> 
> All strands are the same length but one has an overhang.
> 
> I'm curious because of 100bp markers etc. Different companies sell the
> similar ladders but prepared differently i.e. digested plasmids but I'm
> never sure if they are blunt fragments or sticky.
> 
> 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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