HELP: Cloning PCR products-NOT MATT'S!! WRONG ADDRESS!
TedM at darkwing.uoregon.edu
Mon Apr 17 02:51:20 EST 1995
ITS ME TED M. DIDNT CHANGE ADDRESS! UGH SORRY SORRY SORRY..
> Its hard to tell if the enzymes are cutting unless there is a substantial
> piece lopped off.. what are the enzymes? do they cut near ends? NEB has
> some data on this in the back of the catalog. One possible assay involves
> end label and then look for lost signal after cutting. If its not enough
> room you can fill in with T4 pol and then ligate into long concatamers and
> then cut, there is a ref in Methods in Enzymology on this. Of course run
> test ligations and all the relevant controls for transformation.. also try
> to minimize chemical steps for the PCR isolation, my ligations generally
> go better if I have simply cut the band and eluted with a spin column
> (Milipore Millex). Of course get rid of cut ends after digestion with an
> Amicon 30 or equivalent..good luck!
> Ted M.
> Institute of Molecular
Biology, U of O
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