Help: no transformants using BaculoGold

Curt Ashendel ashendel at aclcb.purdue.edu
Mon Jun 6 11:42:50 EST 1994


On 6 Jun 1994 12:55:04 +0100,  <andy.phillips at afrc.ac.uk> wrote:

>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. 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?

The problem also could be with the viral DNA from Pharmingen, but you will 
need a better positive control than your constuct to prove that.  If 
the kit includes some uninserted pVL1392 that you could use as a 
positive control, this will test for problems with technique vs transfer 
plasmids.  It could be either, as quality and quantity of DNA are important 
for the DNA trasfer efficiency, given that the viral DNA is high quality 
and small in quantity.  Technique is important also, and can have some 
affect on transfection (it has more affect on the plaque assay).  The cells 
must be healthy and not too dense when transfected.  Recombianations are 
infrequent events, so I believe it is best to transfect a lot of cells 
(say, in a T25 flask), and let the infection proceed for at least 4 days, 
at which time some visual evidence of focal infections should be evident.

My guess it is that your problem is the quality or quantity of transfer 
plasmid constructs.  My experience with beginners is that this is the most 
common problem at this point in the procedure.

Good luck.

Curt Ashendel
Purdue University
West Lafayette, IN
ashendel at aclcb.purdue.edu



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