Help: no transformants using BaculoGold

ANDY PHILLIPS andy.phillips at afrc.ac.uk
Mon Jun 6 06:55:04 EST 1994


Help!
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

Andy Phillips,
Long Ashton Research Station, Bristol, UK
andy.phillips at bbsrc.ac.uk



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