plasmid re-ligation problem

Austin P. So (Hae Jin) who at what.where
Wed Aug 11 23:48:27 EST 2004


Vagelis wrote:
> However, I encounter a problem during the cloning procedure; the
> double-digested vector re-ligates back to itself, even though the two
> ends are incompatible.

You have to remember that you are not ligating one molecule of plasmid, 
you are ligating trillions of them, so you will get end to end ligations 
occurring:

A---B

A---BB---AA---B...etc...

especially at the concentration of plasmid (1 ug in 20 ul) you are using.

Use much less plasmid (25 ng), and then add 2:1 molar ratio of insert to 
plasmid for your ligation at the volume of 20 ul.

That should be more than enough for your transformations.

> -	1ug double-digested plasmid
> -	1ul  (400 Cohesive End Ligation Units) T4 DNA ligase (NEB)
> -	5ul T4 DNA ligase buffer
> -	up to 20ul with ddH2O
> -	2 hours – room temperature

> The ligated product is loaded on a 1% agarose gel (overnight – 40
> volts). The double-digested vector re-ligates proportionally to the
> amount of T4 DNA ligase. In fact, virtually all of it re-ligates in
> the presence of 4000 units of T4 ligase. Also, when the modified
> 114mer is added to the ligation reaction in high concentrations, it
> tends to inhibit the re-circularisation of the vector.

Of course it is going to...and that is what you want, isn't it?

If you want to be absolutely sure about your ligation, treat your doubly 
digested plasmid with phosphatase so that it cannot self-ligate 
(circularize or concatemerize).

You can relax...there is nothing going "wrong" and there is no 
"contamination"...

Good luck

Austin

BTW...You are overkilling on your purifications...the Qiagen 
purification kit should be more than enough.




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