Gel analysis of ligation product

[Michael Sullivan]

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:

> 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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