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Gel analysis of ligation product

Gel analysis of ligation product - Protocols and Methods Forum

Gel analysis of ligation product - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 11-11-2008, 06:31 PM
Michael Sullivan
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Default Gel analysis of ligation product



Vinay,

My experience is that doing anything with a ligation reaction other
than transforming it is a waste of time. You might get some slight
amount of information from looking at the reaction products compared
to what went into the reaction, but my experience is that what you
see on the gel doesn't look like anything you'd expect corresponding
to your clone of interest. Basically you'll just find out that the
ligase changed what the fragments look like on a gel and what is a
correct clone may only be a minor product in the reaction.

As far as gel purifying and transforming a putative ligation product:
I'd suggest not trying that. All you'll do is expose your product to
additional UV radiation (and possibly making it non-replicable once
you transform) and losing material.

My opinion is that your best bet is to transform and find your clone
later. All told, minipreps and restriction digests are trivial. If
you have trouble recovering a clone, there's probably some
fundamental problem with the cloning strategy or ligase reaction. The
latter is more easily determined by running a control, not analyzing
ligation products.

Hope this helps.

Mike

On Nov 11, 2008, at 4:28 AM, vinay sj wrote:


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