Problem in the ligation - (Apr/06/2005 )

Hi everyone.

These days, i am trying to ligate my target fragment into the YEp vector. But it takes me too much time, and no transformants is screened. I examined the ligase and the system, and found that maybe the fragments i recovered from the gel is not clean or is not suitable for ligation. What can i do? How to recover the fragments in the gel ? The method i used is:
melt the gel ->add equal volume phenol(Tris) ->liquid nitrogen 30min->centrifuge,12000r/min,10min,room temperature->equal volume CHCl3,centrifuge->1/10 volume 3M NaAc, twice the alcohol->-20, 2h. Would you please tell me the efficiency of this approach?

thax.

BoyChina

Hey,

after isolating your DNA-Fragment out of the gel:
Did you ever check the DNA afterwards on a second gel (few µl)? Have you an idea of the amount of your DNA after extraction? Should give you an idea of the efficiancy of your approach.

If you cut your fragements with the right enzymes than they should be suitable for ligation so I wouldn´t worry about that.

Phenol is a critical point as it can be deadly for enzymes. In general "Ligase" is a very "easy-to-handle" enzyme but phenol can be deadly if it is still present in your ligation reaction. So make sure you extract phenol maybe two or three times with chloroform (which may reduce efficiancy but anyway).

Have you checked that your ligation product has its correct size (by gel) and that you transform your bacteria with the right Plasmid-stuff or can´t you get any ligation at all?
If available you can isolate your fragment with a "kit" (Qiagen, Gel extraction kit??), works fine in general.

Hope this is some input
Good luck
Cheers