Help: no transformants using BaculoGold
andy.phillips at afrc.ac.uk
Mon Jun 6 06:55:04 EST 1994
We're trying to express a number of plant cDNAs in insect cells using
a Pharmingen kit. We've cloned our inserts into pVL1392 and tried to
cotransfect Sf21 cells with BaculoGold. We've use both the Ca sulphate method
supplied by Pharmingen, and lipofectin, but have seen no infected cells.
Wild-type virus infects fine, as judged by both plaque assay and polyhedrin
band on SDS-PAGE. This suggests that the cotransfection is going wrong
somewhere. Can anyone with experience of baculoviral problems suggest where we
should look for a cure first?
The two obvious sources of the problem are the cells and the
constructs in the transfer vector. As we have several different constructs, the
latter seems unlikely, unless our plasmid maxipreps are at fault. A contributor
to this bboard suggested that getting healthy cells was the most difficult
aspect for beginners. Is it possible that our cells won't transfect, although
they _infect_ perfectly happily?
Thanks for any help
Long Ashton Research Station, Bristol, UK
andy.phillips at bbsrc.ac.uk
More information about the Methods