Cell biolabs ViraBind Series Product Manual - Page 5

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Preparation of rAAV-2 or rAAV-DJ Samples

The following procedure is suggested for one 15 cm dish or two 10 cm dishes and may be optimized to

suit individual needs. Please refer to the user manual when using Cell Biolabs' AAV Helper-Free

System or other packaging systems.

1. Use HEK 293 cells that have been passaged 2-3 times prior to transfection. Culture these cells

until the monolayer is 70-80% confluent.

2. Cotransfect cells with the pAAV-RC, pHelper and your expression construct according to

manufacturer's manual.

3. After 48-72 hrs, add 0.5 M EDTA to a final of 10 mM to the plate and incubate for 3 min at

room temperature. Gently shake the culture plate several times and harvest all media, including

cells, in a sterile tube.

Note: Trypsin may be substituted for EDTA in this step.

4. Centrifuge for 5 min at 3000 rpm to pellet the transfected cells. Resuspend the cell pellet in 2.5

mL of serum-free DMEM.

5. Subject the cell suspension to four rounds of freeze/thaw cycles by alternating the tubes

between the dry ice-ethanol bath and the 37ºC water bath.

6. Collect the AAV supernatant by centrifugation at 10,000 x g for 10 minutes. Discard the pellet.

7. (Optional) For AAV samples produced with a helper adenovirus, inactivate the helper

adenovirus by incubation the sample at 56ºC for 30 minutes. Collect the AAV supernatant by

centrifugation at 10,000 x g for 10 minutes. Discard the pellet.

8. The viral supernatant can be stored at -80ºC or immediately purified (see purification

instructions below).

Protocol

I. Purification

The following procedure is written for purification and concentration of 2.5 mL of AAV supernatant.

For AAV samples that are less than 2.5 mL, add serum-free DMEM to the final volume of 2.5 mL.

1. Add 25 µL of ViraBind™ AAV Reagent A to 2.5 mL of viral supernatant, mixing well.

2. Incubate for 30 minutes at 37ºC.

3. Centrifuge the AAV supernatant for 15 minutes at 5,000 x g.

4. Carefully collect the supernatant and transfer to a new tube. Discard the pellet.

5. Incubate ViraBind™ AAV Reagent B for 30-60 minutes at 37ºC to ensure Reagent B is

dissolved. Add 125 µL of ViraBind™ AAV Reagent B to the ViraBind™ AAV Reagent A

pretreated 2.5 mL of viral supernatant, mixing well.

6. Incubate for 30 minutes at 37ºC.