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Directional cloning

Directional cloning - Protocols and Methods Forum

Directional cloning - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 03-27-2006, 08:30 AM
newsnet customer
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Default Directional cloning



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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  #2  
Old 03-27-2006, 04:26 PM
Michael Sullivan
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Default Directional cloning

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:


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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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