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[ccp4bb]: Cloning !!!
Hoi,
For the transformation, you should have a positive control ! Try to transform
cells with the original pET-29 vector. Then, you should also check your
enzymes. Again, you can do a control for each enzyme on the original vector
(separated experiments). Put both experiments on gel against markers and
the undigested vector. For each enzyme, you should get one linear fragment.
For the PCR product (i guess you get it from a PCR experiment), it is more
difficult to check. My guess is that you have a problem with the NdeI enzyme
If i recall it correctly, NdeI needs quite quite a lot of bases upstream of the site
to be able to cut (let say it should be more efficient). You should check the catalogue of
the company from which you bought the
enzyme. You should also check your disgested fragment on gel to see if you don't
get some STAR activity by the enzymes.
For theligation experiment, be sure that you have more insert than vector and try the ligation
at room temperature and in the cold room overnight. Again you can check the ligase on
with the vector cut with only one enzyme and then do the transformation. The last possibility
i see could be that you made a mistake for the primers (if PCR) !!!
Good Luck,
Greg
________________________________________________________________
Gregory Verdon
Laboratory of Biophysical Chemistry
Department of Chemistry
University of Groningen
Nijenborgh 4 tel : +31(50)3634384
9747 AG Groningen fax : +31(50)3634800
The Netherlands e-mail: g.verdon@chem.rug.nl
http://www.xray.chem.rug.nl/
________________________________________________________________