jakami at ucdavis.edu
Thu Jun 7 16:30:49 EST 2001
I know you probably thought of these, but some thoughts are:
1). RE sites too close to the ends for efficient cutting. The NEB web site talks about this for some enzymes.
2) Wrong Ligation Buffer. We had a postdoc who accidentally used ligase dilution buffer instead of reaction buffer. No ATP = No ligase
3) Sour Ligase. Ligase is very temp sensitive. 65 is used to inactivate it, but it will degrade at even room temp, albeit more slowly.
4) Competent cells not so competent. Even with the best of vector preps, we always see some background colonies from the vector.
5) Drop Dialysis. You didn't mention if you removed the ligation salts before transformation. Absolutely essential for electroporation,
HIGHLY recommended for chemically competent cells.
Just some ideas, good luck.
Dept. of Agronomy
University of California
Davis, California 95616
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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