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

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Fri Jan 12 04:38:35 EST 2001


In article <200101111630.f0BGUfc20228 at ns.qnis.net>, the eminent Deanne
Bell at BIOSCI/MRC Human Genome Mapping Project Resource Centre wrote
>To Dr. Hiranya S. Roychowdhury"
>I must comment on how polite you are!!  Rather than being so rude, would
>you kindly tell me where my logic is incorrect?
>I made it quite clear that I have not done Restriction fragment gels
>before.  And the original question did not clearly state their puposes.
>D. Bell
>
>----------
>> From: "Dr. Hiranya S. Roychowdhury" <hroychow at nmsu.edu>
>> To: methods at hgmp.mrc.ac.uk
>> Subject: Re: Fw: What is the effect of a 5' overhang on gel mobility?
>> Date: Wednesday, January 10, 2001 6:31 PM
>> 
>> Do you really know what you are talking about?
>> >
>> >
>> >I haven't really done any of this work but i would think that 4 bp would
>> >make a difference.
>> >When you run restriction fragments out on a gel, I think the fragments
>are
>> >denatured and run in such a manner that you have resolution to 1 - 2 bp.
>> >Kind of like a sequencing gel.  In my humble opinion (IMHO) if you want
>> >these two fragments to migrate down a gel at the same speed, you should
>> >make the PCR product as long as the restriction fragment including the
>> >overhang.  Then denature all the fragments and run the gel.
>> >

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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