Transposition Bacmid DNA Mini Culturing baculo cells Transfection of Sf9 cells with recombinant Bacmid DNA Amplification of the virus Titration of baculo stocks Testing the expression
Transposition 1. Plates: 50 µg/ml kanamycin 7 µg/ml gentamycin 10 µg/ml tetracycline 100 µg/ml Bluo-gal 40 µg/ml IPTG 2. Thaw the DH10Bac competent cells 3. Dispense 100 µl of the cells into 15ml tubes 4. Add approximately 1 ng (or little more) recombinant donor plasmid (in 5 µl) and gently mix by tapping the tube 5. Inc. on ice for 30 min 6. Heat shock at 42ºC for 45 sec 7. Chill the mixture on ice for 2 min 8. Add 900 µl SOC to the mixture 9. Shake at 37ºC for 4 h 10.Plate 100 µl of mixture and 100 µl of 1x10-1 & 1x10-2 dilutions 11.Inc. at 37ºC at least for 24 h 12.White colonies contain the recombinant bacmid, restreak the white colonies as short lines to ensure the color 13.Always pick and test several colonies, they may act differently in baculo cells
Culturing baculo cells 1. Pour off the old medium and add 5-10 ml fresh medium. 2. Unattach the cells either by hitting the flask (good for TN5) or by scraping (I like with Sf-9). 3. Mix and add 1/3-1/5 of it to a new flask with 20-30 ml of fresh medium. Close the cap tightly. 4. Let grow for 3-4 days at 28ºC. Medium: Sf-900 II or TNM with or without serum with or without antibiotics (Gibco s Antibiotic and Antimyotic is good, 15240-062) (At the moment Sf-900 with antibiotics works fine.) Filter sterilize
Bacmid DNA Mini 1. Inoculate 2 ml LB+antibiotics o/n cultures. 2. Spin down 1,5 ml of culture 3. Remove the supernatant and resuspend the cells in 0,3 ml of Sol I. Add 0,3 ml of Sol II and gently mix 4. Inc. at RT for 5 min 5. Slowly add 0,3 ml of KAc ph 5.5 mixing gently during addition 6. Inc. on ice for 5-10 min 7. Spin for 10 min at 14K x g 8. Label another Eppendorf and add 0,8 ml of isopropanol to it 9. Gently transfer the supernatant to the tube containing isopropanol. Invert the tube a few times and place on ice for 5-10 min 10.Spin 15 min 14K x g at RT 11.Remove the supernatant and add 0,5 ml of 70% EtOH. Invert a few times 12.Spin 5 min 14K x g at RT 13.Air dry the pellet briefly and dissolve in 40 µl TE 14.Store at -20ºC Solution I 15 mm Tris ph 8 10 mm EDTA 100µg/ml RNase A Solution II 0,2 M NaOH 1 % SDS Antibiotics 50 µg/ml kanamycin 7 µg/ml gentamycin 10 µg/ml tetracyclin
Amplification of the virus For amplifying viral stocks, infect a monolayer culture at MOI of 0,01 to 0,1. Use the following formula: Innoculum required (ml): desired MOI (pfu/ml) x (total nr of cells) titer of viral innoculum (pfu/ml) For example, infect a 50 ml culture at 2 x 10 6 cells/ml with 0,5 ml of a viral stock that is 2 x 10 7 pfu/ml, for a MOI of 0,1. Or: => 175 cm2 flask infected with 0,4 ml of a viral stock diluted with 9 ml of SFM w/ FBS. Incubate 1-2 h at 27 C then 20 ml SFM w/ FBS added. Harvest virus at 48 h post-infection. Centrifuge 1000 x g, 10' at 4 C, and save the supernatant. Store at 4 C. Add FBS to a final conc. of 2% for long term storage, and store an aliquot at -70 C. This will result in approximately 100-fold amplification of the virus. Note! Infecting the cells with too much virus kills the cells!
Titration of baculo stocks 1. Prepare 6 cm Petri dishes with 2 density 1,5x10 6 ja 2x10 6 Sf9 cells per plate(media Sf-900, Antib, FBC). Inc.. 1-2h 27 C. At least 2 plates per dilution is needed. (75 cm 2 flask contains about 35 million cells) 2. Dilute virus stock with Sf-900 (no FBS, antib.): 1x10-6 and 1x10-7 for amplified stocks 1x10-4 - 1x10-6 for primary stocks 1x10-3 - 1x10-7 for unknown titer stocks 3. Wash plates 1x w/ SF-900 media w/o FBS, antib. 4. Cover plates w/ 1 ml of diluted stocks, incubate 1 h RT w/ gentle mixing from time to time 5. Prepare cover agarose (0,5 %, 4 ml per plate). For 50 plates prepare: A: 100 ml 1 % agarose (GTG or other high-quality, but not low melting agarose) in water B:100 ml mix: 80 ml 2xSF-900 (may presipitate at 40 C) 20 ml FBS 0,4 ml Gentamycine, 10 mg/ml 6. Inc. stocks A & B at 42 C. Mix 1:1 before usage. 7. Remove virus stocks from plate and cover plates immediately with 4 ml cover agarose. Keep 1 h RT. 8. Seal the plates w/ parafilm strip, inc. 8-14 days at 27 C 9. Count the plaques
Transfection of Sf9 cells with recombinant Bacmid DNA 1. Seed 1 million cells per 35mm plate in 2 ml Sf-900 +antib. 75cm 2 flask contains about 35 million cells 2. Allow to attach at 27ºC for at least 1 h 3. Prepare the following solutions: A: For each transfection dilute ~5 µl of bacmid DNA into 100 µl Sf-900 w/o FBS&antib. B: For each transfection dilute ~6 µl CellFECTIN into 100 µl Sf-900 w/o FBS&antib. (mix CellFECTIN before use) 4. Combine A & B, mix gently and incubate 15-45 min RT 5. Wash the cells with 2 ml of Sf-900 w/o FBS&antib. 6. Add 0,8 ml of Sf-900 w/o FBS&antib into the A&B mix. Mix gently. 7. Aspirate wash media from the cells and overlay the mix onto the cells 8. Inc. 5 h at 27ºC 9. Remove the transfection mixes and add 2 ml of Sf-900 w/ FBS&antib 10.Inc. 72h at 27ºC 11. Transfer the supernatant to a sterile tube 12.Centrifuge for 5 min at 500 x g and transfer the supertant to a fresh tube 13.Store the virus at 4ºC in dark. For long term storage add FBS to a final conc. of 2 % Sf-900 w/ FBS&antib: Sf-900 10% FBS 0,5 x antib. (50 units/ml penicillin, 50 µg/ml streptomycin)
Testing the expression 1. Seed 3,5 cm or 6 cm plates with 1 or 2 million cells, respectively. 2. Let them attach for 1 h at 27 C. 3. Change the medium. 4. Infect the cells with appropriate titrate or with different volumes, for example 100 µl, 300 µl, 700 µl and 1000 µl. -> 27 C 5. Use some other virus as a (second) control, as the bands may be different from cells with no virus. 6. If you wish you can remove the virus and replace it with fresh medium after 3 hours. 7. Let the cells grow for 24 h or more. 8. Scrape the cells and spin them down, 1000 x g, 10', 4 C, keep on ice form now on. 9. Resuspend the cells in 100-200 µl 20 mm Tris - 200 mm NaCl - PMSF. 10.Add TritonX-100 to a final concentration of 1%. 11.Inc. 15' on ice 12.Spin 3000 rpm, 5' at 4 C. Take the supernatant to a new tube. 13.Add LSB and boil for 2'. 14.Run 20 µl in SDS-PAGE.