Gel analysis of ligation product

Dr. Hiranya S. Roychowdhury via methods%40net.bio.net (by hroychow from nmsu.edu)
Tue Nov 11 12:42:10 EST 2008


In my opinion, gel analysis of ligated products is a step that wastes time
and resources.  Transformation is an overnight process, and individual
colonies can be checked by either a simple PCR or by a quick and dirty
plasmid prep.
Ligation products may or may not show up on a gel, depending upon how the
gel is prepared and run.  Besides, one is simply wasting the ligation
products by running a gel.


> Hello,
>
> I have a question about gel analysis of ligation product. I would like to
> insert a 1.7 Kb fragement into a 3.3 Kb vector. I have done the
> restrictions. I would like to do a gel analysis before transforming to
> save me time with mini preps. I have done around 10 ligations so far. On
> the gel, I see that some times I get blanks(i.e no vector, no insert and
> no ligated products). Some times I get ladders of various combinations of
> vectors and inserts. These ladders do not transform and I do not get any
> colonies. Now, my question is if the blanks are the optimal ligation
> products ? I read in his forum that since ligated products have varying
> winding numbers, they are not to be seen on a gel. Can anyone confirm this
> ?
>
> I know that nicked circles migrate slowly on a gel and I was expecting a
> slowly moving band, which I was planning on purifying and transforming.
> But so far I have not been able to get this.
>
> Any help would be appreciated.
>
> Regards,
> Vinay.
>
>
>
>
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-- 
Hiranya S. Roychowdhury, Ph.D.
Asst. Professor,
Health & Public Services
Dona Ana Community College
New Mexico State University
Las Cruces, NM 88003



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