I am working with the bac-to-bac expression system and following the Invitrogen protocol available online. I have generated my recombinant bacmid and now I should transfect my cells, though they won't transfect. I've been trying for a month now, completely in vain. I use Cellfectin 2 reagent, the same one which is recommended in the protocol, but with no results. Once, however, I infected them with a wild type bacmid, which contained no insert and extensive CPE could be noticed already on the 2nd day. If I check my supernatant with EM I can see the expressed virus particles, but in very low number. Any ideas on what the problem could be? Any other method suggestions that might work well?
Thank you in advance, this is starting to get very overwhelming.
One of the possibilities is that the bacmid prep is not good enough. There are many bacmid prep kits available. However, the most reliable method is home method by isopropanol precipitation. The simplest way is to follow miniprep until to the last step, which you add 1:1 isopropanol and centrifuge down the bacmid, instead of loading it onto filter. Only one thing you need pay attention, which is that after you add lysis buffer, be gentle to the sample as the bacmid is very big, easily to be sheered.
Dear Judith,
Maybe you would like to try an alternative for transfection; I would like to propose you our FlyFectIN transfection reagent, dedicated to insect cells (Ag55, s2, sf9, Mos20, Kc167...). You can have more info in the link below.
this reagent was previously demonstrated to transfect and co-transfect sf9 with DNA by Losfeld et al (2011) and Torres-Vega et al (2014) (respective links: http://www.ncbi.nlm.nih.gov/pubmed/21575138 & http://www.ncbi.nlm.nih.gov/pubmed/25338921.
I will be glad to send you a sample of lullaby; you can contact me via ResearchGate or directly at [email protected]
all the best,
Cedric
http://www.ozbiosciences.com/transfection-cell-specific/28-insect-cells-dna-transfection-flyfectin-reagent.html
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