|Co-transfection of linearized baculovirus DNA with a plasmid transfer vector|
|Note: Any protocols and reagents that you may have used in the past for co-transfection of a plasmid transfer vector with linearized baculovirus DNA (for example BaculoGold™) can be used with ProGreen™, ProEasy™ , ProFold™-C1, ProFold™-C2 and ProFold™-ER1 linearized baculovirus DNA.|
1. Prepare monolayers of insect cells. Monolayers of Sf9 or Sf21 cells at approximately 50-60% confluency (semi-confluent monolayers) should be used for transfections. Higher cell densities are not recommended as cells can rapidly form confluent monolayers. Overgrown monolayers with small crowded cells are incapable of supporting efficient baculovirus replication, and a monolayer that grows to confluency early in the transfection experiment will have the recombinant virus yield dramatically reduced*. However, if the monolayer is too sparse, depending on the medium and the plastic quality, cell replication could be impeded and the virus yield could be reduced also.
Seed monolayers of Sf9 or Sf21 cells to about 50-60% confluency in 8.8 cm2 Nunclone Delta treated Petri dishes (Nunc Cat. #150318) or in 6-well plates. Typical seeding density for 50-60% confluence of Sf9 or Sf21 cells is about 1 x 106 cells/well of a 6-well plate or 8.8 cm2 dish. However, since hemocytometer counts are approximate, it is important to visually ascertain that the the monolayers are indeed at 50-60% confluency. We routinely seed cells from log phase suspension cultures, but cells resuspended from monolayers can be used also.
Cells grown in any media that are recommended for cultivating Sf9 or Sf21 cells can be used. However, TC-100 medium without addition of serum and antibiotics should be used in transfection. After seeding the monolayers, remove the cell culture medium and wash the monolayers twice with 1 ml of sterile PBS or TC-100. Do not operate more than 20 monolayers at once because of the risk of drying the mololayers. Add 1 ml of TC-100 medium without serum and antibiotics to the monolayer. The monolayer is now ready. It should be kept at r.t., and can be used for transfection any time during the same day.
2. Gently mix 0.1 μg of plasmid DNA containing a gene of interest, 5 μl of baculovirus DNA and H2O up to 50 μl.
3. Make 10% Profectin. Dilute only the needed amount of Profectin™ Transfection Reagent 1:10 in sterile deionized water 0 to 1 hour prior to co-transfection. For example, if you are performing one co-transfection, add 5 μl of Profectin™ to a sterile 1.5 ml Eppendorf tube, add 45 μl of water and mix gently by pipetting up and down 2-3 times. Similarly, mix 10 μl of Profectin™ with 90 μl of water, 15 μl with 135 μl, or 20 μl with 180 μl for 2, 3 or 4 co-transfections, respectively. It is also important to avoid creating bubbles in the diluted transfection reagent when mixing. No special tubes are required for diluting the reagent, and any remaining diluted Profectin™ should not be stored or used for future transfections.
4. Add dropwise 50 μl of 10% ProFectin™ from step 3 to 50 μl to the DNA mixture from step 2. Incubate at r.t. for 15-20 min.
5. Add dropwise DNA-Profectin™ emulsion from step 4 to semi-confluent monolayer of insect cells from step 1. The monolayer is in 8.8 cm2 Petri dish containing 1 ml of serum-free and antibiotic free medium TC-100.
6. Incubate at r.t for 12-24h. Add 1 ml of TC-100 medium supplemented with 10% FCS and antibiotics.
7. Incubate at 28°C for another 50-60h, harvest* recombinant baculovirus and study protein expression..
* If by the time of harvest the monolayer is badly overgrown, this usually means that majority of the cells are not infected and the virus titer is low. However, this can be easily amended. Gently resuspend the monolayer using P1000 pipette and seed half of the material into 25 cm2 plug seal cap flask containing 5 ml of complete growth medium with 10% fetal calf serum and antibiotics. Incubate at 28°C for 2-4 days. Initially, uninfected cells will grow and the monolayer will become more confluent. However, the growth will be impeded in 1-3 days, when eventually all the cells in the monolayer become infected. For the best titer, harvest recombinant baculovirus when pronounced cytopathic effect is observed and up to 50% of the cells are dead.
|BaculoGold™ is a trademark of BD Pharmingen.|
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