For the production of baculoviruses we make use of the GIBCO BAC TO
The complete protocol can be downloaded by the web side of GIBCO Life Technologies but is also available from me.
Therefor I will give just a brief overview over the technique used.
The first step to make your personal baculovirus hopefully expressing your favorite gene is to clone it into the correct pFASTBAC HT Vector ( map pFASTBAC HT ), with the standard expression vector construct used in the this will be the HT C. After the cloning is done transform it into DH5a and make a Glycerol stock of the correct clones.
Here comes now the real smart thing about the technique.
First you will have to prepare LB plates containing:
50 mg/ml Kanamycin
7 mg/ml Gentamycin
10 mg/ml Tetracyclin
100 mg/ml Bluo-Gal
40 mg/ml IPTG
If you cot those ready to go, thaw the DH10BAC competent cells (those have to be bought from GIBCO) on ice and dispense them into 15 ml round bottomPP tubes (100ml per tube, but one can reduce this down to twenty without any problem, i did so). Add about one ng donor plasmid this is your construct in the fastbac vector and mix gently. Leave on ice for 30 min. heat shock at 42 C for 45 " and chill on ice for about 2'. Add 900ml of S.O.C. Medium and incubate 37 C with medium agitation for 4 h. GIBCO recommends here a serial dilution, I usually plate simply 10, 100 and 200 m of the transposition culture on the plates, see above. Incubate 24 - 48 h at 37 C until you can discern blue and white colonies, this usually takes at least 36 h.
3. ISOLATION OF THE BACMID DNA
Pick white colonies, they are quite a bit bigger than the blue guys,
and grow them in LB (containing the whole set of antibiotics, see above)
and grow them around 36 hours at 37 C. You may want to restreak the colonies,
this is what is recommended by GIBCO, I found this step obsolete.
Transfer 1.5ml of the stationary culture to an eppi and centrifuge 30" at max speed. remove the supernatant completely(!!) and resuspend the pellet in 300ml Sol. 1 (50mM Glucose, 25 mM Tris-Cl pH 8.0, 10mM EDTApH 8.0, 100mg/ml RNAse A). Add 300 ml Sol. 2 (0.2 N NaOH, 1% SDS) mix gently and incubate 5' at rt. Add 300ml Sol. 3 (3M KAc., pH 5.5) and mix gently. Centrifuge 10' at max speed. in the meantime label another Eppi and put 800 ml isopropanol into it. Pour the the supernatant into the isopropanol without transferring any of the pellet, sometimes a bit of it is actually floating on the top so take care. mix gently and place on ice for 10', spin at 4 C for 30', wash twice with cold 70% Ethanol and air dry for 5' or so, don't overdry or you have a hard time to get your DNA into solution. At 50 ml TE and leave at rt for sometime, donnot pipette up and down. Store at 4 C.
Check for quality by doing a gel analysis 0.5% gel and for insert by PCR.
4. TRANSFECTION OF SF9 CELLS WITH BACMID DNA
Seed 900.000 SF9 cells per 35mm dish in 2 ml of Sf-900 II SFM medium
containing 0.5X Pen/Strep. The cells have to be in good shape and in logarhytmical
growth. Allow them to attach for at least one hour.
While the cells attach you can formulate the transfection complexes: To do so prepare the following solutions in 12X75mm sterile tubes.
1. For each transfection dilute 5 ml of the mini prep bacmid DNA into 100ml Sf-900 II SFM without antibiotics.
2. For each transfection dilute 6 ml Cellfectin into 100ml Sf-900 II SFM without antibiotics. (resuspend the lipid before use)
3. Combine the two solutions by pipetting solution 2 drop wise into solution 1, mix gently don't vortex just tap, and let stand for 15 to 45'.
Meanwhile you can wash the cells once with medium without antibiotics (I am sure pbs will do, but so far I never tried).
For each transfection add 0.8 ml medium to each tube containig the complexes, mix gently and overlay the cells with diluted lipid DNA complexes. Rock slightly but don't swirl and but them back into the incubator. leave them there undisturbed for 5h. After this incubation period exchange the media with 2 ml media containing antibiotics. Harvest the virus after 72 h post transfection.
It is a good idea to harvest the cells to and analyze them on a SDS gel for expression (western).