Biology Reference
In-Depth Information
8. The pressure should not exceed the maximum suggested by
the manufacturer. At least 3 L of CAV-2 vector stocks can be
filtered using 500 cm
2
membrane capsule filters with little
backpressure [
16
].
9. Expected recovery of CAV-2 in the clarified sample using this
method is high (84 %,
n
= 3) as determined by real-time PCR
(qPCR) [
16
,
19
].
10. The hollow fiber cartridge should always be connected in the
same direction. Draw arrows to indicate flow direction the first
time it is used. Secure all connections. Tubing can be reused.
However, it is recommended to replace the tubing connected
to the cartridge inlet every two or three runs since it passes
through the pump and there is risk for damage. Check all sys-
tem tubing to ensure it is not kinked or pinched.
11. Alcohol enhances glycerol removal. The inlet (Pi) and outlet
pressures (Po) should be low at this point. The permeate pres-
sure (Pp) is ~0 throughout the process.
12. Excessive TMP can cause premature membrane fouling (gel
layer effect). Thus, it is important to adjust filtration parame-
ters in early experiments. The optimal TMP is achieved by
gradually increasing the TMP by pressing the backpressure
valve while monitoring the flow rate. Once the permeate flow
rate does not increase by further pressing the valve, the desir-
able TMP has been reached. Using the Midjet
®
and Quixstand
®
,
this flow rate was around 1.6 and 11 mL/min, respectively
(~60 LMH for both) and the TMP around 1.5 bar or 25 psi,
respectively, for our particular CAV-2 feed [
16
]. Feed volumes
of 0.2 L and 1 L could easily be processed using a hollow fiber
cartridge with a membrane area of 16 and 110 cm
2
,
respectively.
Equations:
(
)
Pi Po
Pp
+
(
)
=
Transmembranepressure TMP
2
−
(
)
×
Flow mL min
Membranearea
/
006
2
(
)
=
RecirculationflowrateLMH
(
)
m
13. High recovery of CAV-2 in the concentrated and benzonase
treated samples is attained (98 %,
n
= 2 and 83 %,
n
= 3) as
determined by qPCR using the Midjet
®
and Quixstand sys-
tems, respectively. A noteworthy removal of contaminating
proteins is also accomplished (55 %,
n
= 2 and 43 %,
n
= 3) [
16
].
14. The commercial benzonase
®
stock is typically supplied at a
concentration of 250,000 U/mL. To reach a concentration of
10,000 U/mL in the working solution (WS), the original
