Biology Reference
In-Depth Information
3. Set up the filtration system in the biological safety cabinet (
see
Note 7
).
4. Filter viral stocks at constant flow rate (start at 10 mL/min and
gradually increase up to 50 mL/min). Collect the permeate in a
sterile collection bottle. Check pressure at all times (
see
Note 8
).
5. Measure the final volume and aliquot clarified virus stock sam-
ples for analyses (
see
Note 9
).
6. Keep the virus stock at 4 °C until the next downstream pro-
cessing step or store at −80 °C along with the aliquoted
samples.
1. Set up the ultrafiltration system in the biological safety cabinet
(
see
Note 10
).
2. Recirculate 200 mL of 20 % EtOH through new cartridges
during 10 min to remove trace amounts of glycerol humec-
tants. Then clean cartridges by recirculating with 200 mL
Milli-Q H
2
O during 15 min and flush the cartridge membrane
with 200 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 reservoir (max. volume
200 mL). Aliquot starting material samples for analyses and
measure the starting volume.
4. Connect the feed reservoir and start pumping sample through
the cartridge. Adjust the transmembrane pressure (TMP) to
attain a recirculation flow of 60 liters per square meter of mem-
brane area per hour (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 third
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
)
3.2 Concentration
by Cross-Flow
Ultrafiltration-
Diafiltration
3.2.1 Midjet
®
System
(Up to 200 mL)
