plasmid re-ligation problem

Ferdinando Pucci brotizolam at libero.it
Wed Aug 11 14:35:33 EST 2004


On 11 Aug 2004 11:48:01 -0700
el36 at le.ac.uk (Vagelis) wrote:

> 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.

How did you realize that is the vector recircularized? Did you see a
band a little under the supercoiled wt vector or you have another
plasmid the same size?

> The same problem occurs even when I perform double digestions or when
> I do not purify the digested vector with agarose electrophoresis. I
> also dismissed the possibility of contamination in my ligation buffer
> or in the T4 ligase.
>  
> My guess is that there is either a misdigestion problem during the
> second digestion or that the 114mer is indeed excised but not
> separated from the rest of the vector.

I'm not a PhD nor a MD but i'm working with this to get a degree, so i
can try to say something not so strange :-) imho probably there is a
degradation problem of your stickies that become blunt and... I would
try to change the purification kit or to have more care with hazardous
passages.

Good work!

PS: i'm used with so much RE as 1U/ug of DNA but you beat me! :-)
PPS: maybe with a conc of 50ng/ul in the ligation reaction there could
be too much probability of self ligation, you should calculate it
(search previous posts here)



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