1. Remove all but 2 ml per well of medium from  6-well plates containing 80% to 90% confluent 911 or 293 cells.  Infect with appropriately diluted virus for 3 to 6 hours.  Infect cells with 6 different dilution titers (e.g. 10^-3 to 10^-8ml/well).

2. Prepare the overlay agar as following:  autoclave 100 ml of 2.8% Bacto-Agar (Difco) and keep warm at 45^oC water bath. For 100 ml overlay:

                                              Stock Conc.      Final Conc.         ml/100ml overlay

2 X BME (GIBCO)                       2 X                  1X                         50.0
HEPES                                       1.0M             20.0mM                      2.0
MgCl2                                         1.0M             12.5mM                     1.25
FBS                                          100.0%             10.0%                      10.0
Pen/Strep                                  100 X                  1 X                         1.0
                                       mix well and incubate at 37^oC bath.
Bacto-Agar                             2.8%                   1.0%                      36.0

             Mix well and swirl at 37^oC water bath, add 4 ml/well for 6-well plate.

Note:  If you want to melt 2.8% agar stock,  microwave or heat it in boiling water bath.  Prepare 50 ml overlay at a time. To prevent the pre-drying of agar before plaque formation, it is important to add sterile PBS or Hank’s medium in the space between wells, and wrap the plates with SaraWrap.

3. Return the plates to 37^oC CO₂ incubator.  On days 5 - 7, overlay 2 to 3 ml agar containing nuetral red (from 100x stock, available from GIBCO-BRL) to each well.  Plaques should be visible 16-30hrs after the neutral red overlay. (In general, plaques should form in 911 cells in 4 or 5 days and in 293 cells after 5-7 days).

4. Pick 5 to 10 well-isolated plaques for each recombinant by punching out agar plugs with a sterile pasteur pipet. Store agar plugs in 200µl HBSS medium. Perform freeze/thaw/vortex cycle 4 times. Spin samples briefly.

5. Infect one well of 6-well plate of 911 or 293 cells (about 80-90% confluency at time of infection) with 50-70% virus sup of each plaque for 3 to 5 days (depending on the titer of individual plaque). Presence of recombinant virus can be confirmed by Western blot or PCR.

6. Collect viral supernatant using four cycles of freezing/thawing, as described earlier in this guide. The virus sup can be used to further infect T-25, and then T-75 flasks, for large scale preparation.

D.  CsCl Banding of Plasmid DNA

1. Collect  400 ml cultures (from one 1L flask) in 500 ml centrifuge containers in Sorvall GS3 rotor (holds up to six 500 ml containers). Spin @ 6k (for 10 min @ 4^oC in Sorvall  centrifuge.

Alternative:  use 250 ml conical tubes, cf. 3000 RPM (2500 g) in IEC at 4 degrees for 15 minutes and follow "Wizard Protocol" for alkaline lysis and isopropanol
precipitation steps..

2. Resuspend pellets in 7.5 ml BDI (50 mM glucose, 25 mM Tris pH 8.0, 10 mM EDTA stored @ 4 oC; made as follows: 4.5 gm glucose, 12.5 ml 1 M Tris pH 8.5, 25 ml 0.2M EDTA, 460 ml H₂O, pH 8.05).

3. Transfer resuspended pellets to 50ml polypropylene tubes. Add 2.5 ml 20 mg/ml lysozyme in BDI. Vortex and sit @ RT for 5 min.

4.Add 20 ml BDII (made fresh) (= 0.125N NaOH/1% SDS; make this by combining 1 ml 50% NaOH, 10 ml 10%SDS, and 89 ml H₂O). Invert tubes gently and sit on ice for 10 min.

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