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Bacmid generation

1) Transform chemically competent EMBacY cells with 5 μL of miniprep plasmid (30 min on ice,
then heat shock 42C - 90 sec).
2) Transfer recovered cells to a glass culture tube. Cells recovered in eppis will not have sufficient
aeration.
Leave cells to recover, at 37ºC for at least 6 hrs (For larger constructs (i.e. 4 or more subunits),
an overnight recovery may be necessary.)
3) Plate out on plates containing kanamycin, tetracyclin, gentamycin, X-Gal, IPTG.
4) Leave plates in 37ºC incubator for approx. 48 hrs to allow for blue-white screening to occur.
5) Pick the white colonies and grow 3 mL cultures (LB+Kan+Gen) overnight at 37ºC.
6) Harvest cultures and do the first three steps of the miniprep kit (resuspension in P1, lysis in P2,
clearance in P3 or N3). Do not vortex the cells as this will shear the bacmid!
7) Rather than applying the cleared supernatant to the tube, transfer 700 μL to a 1.5 mL eppi and
add an equal volume of 100% isopropanol. Precipitate either on ice or in the -20ºC (>1 hr, but
can be overnight).
8) Pellet the precipitated DNA by centrifugation at max RPM for 15 mintues at 4ºC.
9) Remove supernatant from the pellet and add 700 μL of 70 % EtOH then spin again for 5 minutes
at max RPM.
10) Remove supernatant and repeat step 9.
11) Relocate to the hood in the insect cell culture. Remove the 70% EtOH first with a P1000, and
then the residual with a P10 – be careful not to disturb the pellet!
12) Leave the pellets to dry under the hood (with the hood left on of course!) this will take about
an hour (if you remembered to use 1.5 mL eppis, and took enough EtOH off with the P10).
13) Resuspend in 30μL sterile TE or sterile H2O. Do not pipette up and down (this might again
shear the bacmid), but simply tap the tube gently.

Transfection
Prior to transfection, Sf9 cells should have been split to 1x106/mL 24 hours before.
1) Make transfection mix (per reaction)
a. 250μL medium
b. 5 μL transfection reagent
c. 10-25 μL bacmid DNA (in either water or TE)
2) Leave transfection mix for 20-30 minutes
3) Count cells (should be around 2x106/mL)
4) Add a suitable volume of cells to a falcon (see example below - 6)
5) Spin cells 5 min at 800 rpm
6) Pour off supernatant and resuspend – carefully! – in a volume of medium, to bring
concentration to 1x106/mL.
a. E.g. If I’m doing 10 transfections, I will need 2 mL of cells at 1x106/mL per transfection.
In this case I would take 12 mL of cells at 2x106/mL and spin. I would then resuspend in
24 mL of medium. I then have 10 transfections, one –ve control and 2 mL of spare cells.
7) Transfer 2 mL of resuspended cells to each well of a 6 well plate.
8) Add transfection mix to each well
9) Leave transfections at 27ºC for 2-3 days (e.g. either Friday to Monday, or Tuesday to
Friday)
10) Examine the cells under the microscope. The negative control cells should be dense, and of a
uniform size. Transfected cells should be swollen, with evidence of lysed cells. Check for
contamination.

Viral amplification
1) Amplification
a. Add 10 mL of freshly resuspended Sf9 cells (1x106/mL) to each 10 cm dish
b. Add 2 mL from 6 well transfection plate (also carry over the –ve control!)
c. Leave for 2-3 days
2) Check the cells under the microscope. There should now be a clear difference between the
transfected cells and the –ve control.
3) Resuspend cells from the 10 cm dish – this might require some patience.
4) Spin resuspension in 15 mL falcon tubes (800 rpm, 5 min).
5) Take off and filter the supernatant into 15 ml tubes (label the tubes with ethanol resistant
marker) – this is our V0. The V0 can be stored indefinitely.
6) With the cell pellets, firstly check the fluoresence of the pellet using the ChemiDoc system.
Place the falcons with the pellets on the UV tray. Start a new single channel experiment, and
choose blots from the menu. Under blots choose ‘fluorescin’ (this has an excitation and emission
spectra similar to YFP). The –ve control should have no fluorescence, the transfected pellets
should be bright. For any sample not showing any fluorescence, discard both the pellet and the
V0.
7) If your construct has an affinity tag, or if you have an antibody that could be used to detecting
an untagged protein, then keep the cell pellets (freeze in liquid nitrogen, and store at -80ºC). Test
the expression of your protein by pull-down assay. This is the most important experiment as it
will give you a clear indication of whether you should proceed with viral amplification and
expression trials. It is of course good practice to proceed with the V1 amplification while doing
these experiments.

Viral amplification – V1 and V2


I usually do two rounds of amplification, to generate a V1 and then a V2. This follows the same
principle as the amplification above; fresh log phase cells at 1x106/mL. In a liquid culture I make
a 1:100 dilution to amplify new virus. The first virus after the V0 I call V1 and the second V2. I use
the V2 virus for expression trials.