Reagents:

• Polyethylene glycol         Sigma, MW10000

• NaCl                              tissue culture grade (?)

• EDTA                            tissue culture grade precipitate (NaCl)

• H₂O                               deionized pyrogen free

• 0.1N NaOH                   pyrogen free

Solutions:

Add 100g PEG and 6g NaCl to 250ml H₂O in 500ml bottle. Mix. Autoclave at 15lb for 20 minutes. Allow to cool. Adjust pH with NaOH to pH 7.2 under sterile conditions.

Protocol:

1.            Infect cells and allow to incubate for as long as necessary to approach maximum virus production for the strain and cell type combination.

2.            Centrifuge culture at 1000rpm for 7 minutes.

3.            Resuspend cell pellet in fresh media. Incubate for an additional 48hrs.

4.            Centrifuge culture at 14000rpm for 15 minutes to remove cells.

5.            Collect supernatant and add 1 volume of PEG solution to 4 volumes of virus containing supernatant.

6.            Refrigerate overnight (stable up to 2 weeks @ 4 °C).

7.            Centrifuge at 2300rpm for 45 minutes.

8.            Remove supernatant.

9.            Resuspend viruspellet in 1/10 to 1/100 of original volume.

10.        Aliquot in small aliquots and store at -70 °C until use.

Procedure gives 1 to 2 log increase in final infectious virus titer.

For specifics on appropriate handling and waste procedures please see the online chemical SOPs or our waste and spill notebook located in room 352.

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