4 replies to this topic

[sandie]

Hi

I am having real trouble getting a couple of ligations to work and have been searching around for any protocols that may help me out.  I have come across a protocol for in gel ligation, where the RE digested insert and vector are run on a low melting point gel, the fragments are excised and melted and used directly in the ligation reaction

Has anyone used this method before?  Does it work?  Are there any special considerations such as liagse type i have to think about?  Does this method work better for troubled ligations than the standard method?

Cheers for any help  

Edited by sandie, 23 February 2005 - 06:15 PM.

[natic]

Hi.
I have used low melting point agarose for DNA purification. after running on gel I excised the fragment and used AgarACE enzyme (promega) that digested the gell. then, used it for regular ligation. the benefit of this method is that the DNA yields are much higher.

[neuromatt]

I've used agarACE as well. However, after digestion I ethanol precipitate so I can get small volumes, right concentrations etc. I usually get +70% recovery and the ligiations work fine.

[henryjekyl]

For trouble ligations I find that the Bioline Quick stick ligase work well. Also if I have a PCR product that has been amplified using linkers, digesting both the vector and the PCR product  in the same tube works to prevent vector self ligation and enhances the normal ligation reaction. If you want me to elaborate on that I can.

[elphyn]

I have done in gel ligations before, about 5 years ago. Not sure which brand of T4 ligase i used but one was better than the other (out of NEB and Promega). I'll see if I can find out.
Ligating in gel helps keep the components together, (this is what I was told) therefore increasing the chances of successful ligation.
I put together several plasmids using this method.

cheers