sergioal at [NOSPAM].es
Tue May 22 03:24:11 EST 2001
lethal (or highly toxic) gene into the insert? -> Try reverse orientation cloning.
did you perform a ligase positive control?. Maybe the problem is in the ligase reaction.
We had some bad experiences using NotI (besides its price), and i don't know how good it is on DNA ends. Maybe you could try to do a
blunt end ligation (refilling the ends or using another polymerase) and get the HindIII-NotI fragment afterwards....
Malathi Hari wrote:
> 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 :)
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