2.6.1 Materials
for the Quantification
of Baculoviruses Using
qPCR with SYBR Green
1. Kit for the isolation of baculoviral DNA (e.g., PureLink
® viral
RNA/DNA Mini Kit, Thermo Fisher Scientific, Waltham,
MA, USA).
2. qPCR Kit (e.g., QuantiNova™ SYBR
® Green, QIAGEN,
Venlo, Netherlands).
3. Suitable Primer for the detection of a specific gene sequence of
baculoviral DNA.
4. Crushed ice.
5. Sterile single-use filter tips (e.g., Biosphere
® filter tips, Sarstedt, Nu ¨mbrecht, Germany).
6. Piston pipettes (e.g., Research Plus, Eppendorf, Hamburg,
Germany).
7. 1.5-mL DNA Low Bind centrifuge tubes (e.g., Eppendorf,
Hamburg, Germany).
8. PCR tubes (e.g., Multiply
®
-Pro 0.2 mL (PP), Sarstedt, Nu ¨mbrecht, Germany).
9. PCR cooler (e.g., Eppendorf, Hamburg, Germany).
10. 96-well plate, semi skirted (e.g., twin.tec. real-time PCR Plate
96, Eppendorf, Hamburg, Germany).
11. PCR film (e.g., Masterclear real-time PCR film, Eppendorf,
Hamburg, Germany).
12. qPCR System (e.g., Mastercycler
® ep gradient S realplex,
Eppendorf, Hamburg, Germany).
13. Multifuge (e.g., Multifuge X1R, Thermo Fisher Scientific,
Waltham, MA, USA).
14. Centrifuge (e.g., Sigma 1-16K, Sigma-Aldrich, Hamburg,
Germany).
15. Thermomixer (e.g., HLC Cooling-Thermomixer, DITABIS,
Pforzheim, Germany).
16. Vortexer
(e.g.,
Lab
dancer,
VWR,
Radnor,
Pennsylvania, USA).
2.6.2 Materials
for Quantification
of Baculoviruses Using
a Viral Plaque Assay
1. Spodoptera frugiperda cell suspension with a cell density of
1.0 Â 10
6 cells/mL (e.g., Sf-9 Insect Cells, Merck, Darmstadt,
Germany).
2. Insect cell culture medium (e.g., Sf900 II, Thermo Fisher
Scientific, Waltham, MA, USA).
3. Generated P2 virus stock.
4. 4% agarose Gel (e.g., Plaque agarose, Biozym Scientific, Hessisch Oldendorf, Germany).
5. Neutral red, high purity (e.g., Carl Roth, Karlsruhe, Germany).
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