Do you also incubate after mixing bacmid-virus genomic DNA and transfection reagent? A half an hour incubation after mixing makes the efficient DNA-liposome complex. 

For transfection in insect cells, Cellfectin or Cellfectin II reagent is commonly used. We do transfection using Cellfectin II and it works fine.

Also, it may depend on your bacmid. If you haven't verify your bacmid, I would recommend to verify it by PCR before proceeding for transfection.

Do you also incubate after mixing bacmid-virus genomic DNA and transfection reagent? A half an hour incubation after mixing makes the efficient DNA-liposome complex. 

For transfection in insect cells, Cellfectin or Cellfectin II reagent is commonly used. We do transfection using Cellfectin II and it works fine.

Also, it may depend on your bacmid. If you haven't verify your bacmid, I would recommend to verify it by PCR before proceeding for transfection.

I incubate the mix for 20 min following the instructions of the kit manual. Also, the FlashBAC kit is theoretically optimized for insect cells, my flashfectin might be quite similar molecule to the Cellfectin. The vector is sequenced and confirmed.

if your concern is the transfection it seems you need to do a control transfection. Do you have any plasmids with insect promoters in them that you could transfect, like Invitrogen's pIZT? you might also try setting up a panel of DNAS concentrations vs transfection reagent; we used this to optimize for different plasmids the transferrin protocols for PEI. Beyond that, are you doing the typical post-transfection treatments like gentle rocking (not orbital).

I agree with titrating the amount of bacmid used. I always had problems with yield when purifying high molecular weight plasmids using Qiagen maxiprep kits. The solution (according to Qiagen) was to elute with pre-warmed elution buffer to release the bacmid from the column. Also, you can check your bacmid using pulse field gel electrophoresis. Maybe most of your bacmid is nicked or denatured and therefore inefficient at transfection (which seems to prefer intact double-stranded DNA). 

Are you sure your transfection reagent hasn't gone off/expired? A couple of years ago I ran into problems getting transfections to work (I had a visual marker of a YFP protein to check for transfection) and it turned out my Cellfectin II reagent had got too old.

What I ended up doing was to simply use PEI - polyethyleneimine - as the transfetion reagent. This has the benefit that it costs next to nothing (

Thanks for the suggestions. I will re-check the concentration of my transfer vector DNA, and play a bit with different DNA-to-flashfectin ratios. I also transfected with a control vector (same pAcUW, same tags with different foreign insert) and failed, and also tested expression by WB in cells after transfection with the initial transfection mix after several days, and had no protein (just to test if I had some transfection yet low virus titer and thus lack of visible hedra...). On top of all these I tried lipofectamine (that is effective even in primary cells in our lab) and all of these conditions in both Sf9 and Sf21 cells and still no success...

Dear Andras,

These insects cell, cf9 and cf21 are sometimes finicky and acting up and do not respond to conventional transfection strategies. In my experience several routes should be undertaken. Chief among them using a low tech transfection mix using different concentration of PEI and DMSO. You should read the literature to see the pore sizes generated in SF9 or sf21 using 5 to 10% DMSO which opens up the pores of the cells and make the construct entry easier. You should also paying close attention to the mw of your PEI used and again use the literature to get a handle on that. Believe it or not, sometime old technologies presented in Maniatis book of molecular biology techniques are still working and in some case are superior.

Good luck,

Chris

Thanks Chris, I will take a look at it.

Dear Andras, Have you solved your problem with the Sf9 cells?  We had the same problem with RNA but found that a high concentration of RNA was precipitating during the transfection. I would be interested in what you have done.

Dear Raquel and Dear All,

Yes problem solved. I have changed 3 things:

1) I prepared Midi-prep (Qiagen) from my transfer vector, it has a double precipitation step in the protocol and produces a cleaner DNA prep (vs mini-prep).

2) I changed my plate!! sounds stupid, but I think it actually solved my problem: I turned to another type of adherent culture plate which held my cells more tightly bound during liquid changes.

3) I turned to another transfection reagent and use Fugene HD.

With these changes I immediately produced viruses, but again, this is true for my transfection system (OET), others might be sensitive for different stuff.

Good luck with the experiments.

Bests, Andras

Dear Szollosi and all,did anyone successfully transfect the sf9 cells with Lipofectimine 2000,I have did three times but all failed.Thanks.  

Hola Andras. Sorry but It´s the first time that I see baculovirus related questions.I hope you have solved your transfection problem, but for future new transfections It¨easier and cheaper use polietilenimine reagent.JetPei. You can transfect with serum in the medium and in my hands never failed. Look information and prices about it if you are intersted. Buena suerte

Dear Andras,

I hope you will get to see this message. Please which plates did you end up using? And by the way, did you ever attempt using DMSO in transfection?