Biology Reference
In-Depth Information
10. Store concentrated samples at −80 °C along with the previ-
ously aliquoted samples.
11. Clean cartridge by recirculating NaOH 0.5 M during 20 min.
Then neutralize with 200 mL of buffer A and 200 mL of
Milli-Q H 2 O. Store in 20 % EtOH at 4 °C.
1. Set up the ultrafiltration system in the biological safety cabinet
( see Note 10 ).
2. Recirculate 500 mL of 20 % EtOH through new cartridges
during 10 min to remove trace amounts of glycerol humec-
tants. Then clean cartridges by recirculating with 500 mL
Milli-Q H 2 O during 15 min and flush the cartridge membrane
with 500 mL of diafiltration buffer A. The membrane should
be kept wet at all times from this point on ( see Note 11 ).
3. Transfer the virus stock into a clean autoclavable reservoir
(max. volume 1,000 mL). Connect the system to a secondary
feed reservoir if volumes larger than 1 L are being processed.
Aliquot starting material samples for analyses and measure the
starting volume.
4. Start pumping sample at 400 rpm through the cartridge.
Adjust the transmembrane pressure (TMP) to attain a recircu-
lation flow of 60 LMH using the backpressure valve attached
to the outlet tubing ( see Note 12 ).
5. Once the volume is reduced by tenfold, empty tubing in order
to collect the entire sample in the feed reservoir. This can be
accomplished by disconnecting the inlet tubing from the reser-
voir cap.
6. Follow the procedure described in Subheading 3.3 to digest
nucleic acids inside the feed reservoir in between the ultrafiltra-
tion and diafiltration operations.
7. Diafilter the sample by adding into the feed reservoir a volume
of diafiltration buffer equal to that of the retentate. Repeat
diafiltration three times in discontinuous mode.
8. The ultrafiltration-diafiltration process is stopped once the
final desired volume of retentate is reached after the 3rd
diafiltration step. Empty the tubing in order to collect the
entire sample in the feed reservoir.
9. Measure the final volume and aliquot concentrated virus stock
samples for analyses ( see Note 13 ).
10. Store concentrated samples at −80 °C along with the previ-
ously aliquoted samples.
11. Clean cartridge by recirculating NaOH 0.5 M during 20 min.
Then neutralize with 200 mL of buffer A and 200 mL of
Milli-Q H 2 O. Store in 20 % EtOH at 4 °C.
3.2.2 Quixstand ® System
(Up to 10 L)
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