ligation problem

NJ njuni at poppy.ocn.ne.jp
Wed May 23 00:30:21 EST 2001


Another possibility is a bad influence of UV applied when you retrieve DNA
from gel. Even when excised DNA fragments seemed not to be degraded as
analyzed by EP, this kind of problem happened to may people in our lab in
the past. I suppose this is due to pryrimidine dimer formation by UV, which
recA- hosts can not repair (I am not sure...). Reduce the dose of UV
irradiation (longer wavelengths would be better), expose only a minimum
strip of lane to observe with masking, or adopt another procedure which does
not require DNA purification from gel.
--
NJ

in article 9ec7lf$fv1$1 at mozo.cc.purdue.edu, Malathi Hari at
mhari at expert.cc.purdue.edu wrote on 5/22/01 8:17 AM:

> Howsoever I hate it, but I have to admit that I am unable to subclone a PCR
> product into my vector.  Here are the specs :
> PCR 1.5 Kb insert from a plasmid using Taq (RE sites internal to the primers
> so no question about sites missing)
> Purify using QIAGEN PCR purification kit
> Digest with HindIII and NotI(or SpeI)
> Heat denature enzymes and ppt. DNA (tried both gel purification or without)
> 
> Vector digested with HindIII and NotI (or XbaI)
> Run on gel to remove an insert already in the original vector (also confirms
> digestion with both enzymes)
> Agarose band spun in a Sigma spin column and DNA ppted.
> 
> Checked purity and yield of vecotr and insert by running on gel and ligation
> setup with excess insert over vecotr at 15C for 24 hrs.
> 
> Control ligations include vector with ligase and no insert
> 
> 
> 
> RESULT : NO CLONES
> 
> I hope u have some ideas :)
> 
> Thanks
> 
> Malathi
> 




More information about the Methods mailing list