Directional cloning

Michael Sullivan mlsulliv at wisc.edu
Mon Mar 27 11:26:22 EST 2006


My opinion is that it would be a waste of time and reagents. Athhough  
it is theoretically possible to have to two vector molecules ligate  
to each other, under the conditions most use for ligation (e.g.  
excess insert) it should be less likely to happen than getting your  
desired clone.  Also, I think creation of a molecule with two similar  
origins of replication might be somehow disfavored (my evidence for  
this is that when I "shotgun clone" an insert from a plasmid [i.e.  
just use the whole digestion of a plasmid as the "insert"] into a  
second vector with different antibiotic resistance, I recover clones  
consisting of both vector pieces [i.e. having both antibiotic  
resistances] less than 5% of the time.

What I consider a fairly "normal" ligation reaction would be

10-50 ng of vector
3 fold molar excess of insert
in a total volume of 5 to 10 ul

One thing that may be happening to give you lots of clones with no  
insert is that the vector digestion is incomplete and there are a  
significant number of vector molecules that are only singly digested.  
Religation of a singly digested vector is VERY efficient, so even a  
small amount of single cut vector can overwhelm finding the desired  
cloning event . If you're vector has color selection, you might be  
able to get around this since religations products would produce blue  
colonies.

Mike Sullivan


On Mar 27, 2006, at 2:30 AM, newsnet customer wrote:

> Hi,
>
> I performed directional cloning of a gene into a vector.
>
> I was wondering if you need to de-phoshorylate the ends even with  
> directional cloning where the cut vector does not have compatible  
> ends?
>
> It would seem possible that a cut vector could ligate with another  
> cut vector (not self ligate), so it would be a good idea to de- 
> phoshorylate the ends when doing directional cloning?
>
> What are peoples opinion? I just screen about 10 colonies and they  
> are all negative, so there must be some form of re-ligation happening.
>
> Cheers,
> Sharp
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---
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
ARS-USDA
1925 Linden Drive West
Madison, WI 53706
(608) 890-0046 (Phone)
(608) 890-0076 (FAX)



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