3 replies to this topic

[Raygoza]

Hi, i;m faced with the fact that highly competent cells would take unligated plasmid and "fix it", so i was contemplating gel extraction but then the resulting amount of dna is too low or very close to nothing. could i simply get the band from the gel, melt it, and then use that to transform the bacteria?

 

 

[bob1]

I don't think they will ligate the DNA themselves, but they can repair single strand breaks. You should be able to melt the gel and use that to transform if you have some DNA that has single strand breaks but is still circular.  I would suggest using low melting point agarose for this - too hot an aliquot of gel will kill the bacteria.

 

It is also possible to elute DNA from gels by spinning the liquid out of the slice

[Trof]

I was recently looking up some things and found these links about in-gel ligation.

 

http://labs.mcdb.lsa..._ligations.html
http://openwetware.o...In-gel_ligation

 

Maybe you'll find some information there useful. It's about ligation, but uses the gel slices without purification to transform.

 

I don't understand however, why do you need to put it on the gel in the first place, if you need bacteria to recircularize (?) a plazmid. Only thing I could imagine, that you need to cut some part out and recircularize the rest, but that would probably happen in a high probability even if you do add ligase. 

[Rajpoot]

just use more DNA e.g 5 microgram for digestion/ gel extraction and elute in less volume of elution buffer e.g. 20-30 microliter. You will get enough DNA (30-50 ng/ul) to be used for ligation and subsequent transformation.