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

Deanne Bell dbell at qnis.net
Wed Jan 10 11:09:04 EST 2001


> "WILLIS GAVIN (RM1) Norfolk and Norwich NHS Trust" wrote:
> 
> > I want to make a (blunt ended) PCR product that will migrate at the
same
> > speed as a restriction fragment with one blunt end and a 4 b.p. 5'
overhang.
> > Should the blunt PCR product be as long as the fragment including the
length
> > of the overhang, not including it or in between?


> From: Susanne Rohrer <"srohrer(removethis)"@immv.unizh.ch>
> What are you trying to do with this?
> Theoretically speaking, unless your fragment is only about 20bp, it would
> probably not matter. But maybe I am missing your point. 
> Susanne



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.

Deanne Bell
Molecular Markers Lab Technician
USDA Agricultural Research Service
2021 S. Peach Ave.
Fresno, CA 93727
e-mail: dbell at qnis.net
phone: 559 453-3170
fax: 559 453-3088


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