Baculovirus Quantitative Titration Kit

* Add Proteinase K to Virus Lysis Buffer before use according to the number of your samples.

† Compatible to ABI Prism 7500/7500 Fast, QuantStudio 3/5/6 Flex/7 Flex System, ViiA^TM 7 System,

Stratagene Mx3000/Mx3005P, Corbett Rotor Gene 3000, et. al.

Additional Materials Required

• Quantitative real-time PCR Thermocycler
• Optical 96-well PCR plates or 8-well PCR strips with strip caps.
• Centrifuge for tubes, PCR strips or PCR plates.

Baculoviral Titration Protocol

1. Centrifuge the medium from baculovirus-infected cells at 500g for 5mins. Transfer supernatant to a new 1.5 mL tube, discard cells and debris.

2. Dilute Virus Standard serially using the RNase-free water to generate standard serial log dilutions containing 6 points, 2*10⁸pfu/mL, 2*10⁷pfu/mL, 2*10⁶pfu/mL, 2*10⁵pfu/mL, 2*10⁴pfu/mL, 2*10³pfu/mL.

3. Dilute your unknown samples, if you suspect the titer to be great than 10⁸pfu/mL.

4. Take 80μL standard serial dilutions and your unknown diluted (e.g., 1 in 5 or 1 in 10) or undiluted samples to PCR tubes, respectively. Centrifuge at 13000rpm for 5min, and then discard supernatant.

5. Re-suspend the virus pellet in 20μL Virus Lysis Buffer (add final 1x Proteinase K before use)in tips.

6. Run the following lysis program to extract virus DNA. The lysis can be used as templates for qPCR.

7. Prepare qPCR reactions on ice. Multiply the following amounts by the number of reactions including 6 standard serial dilutions, unknown samples, 1*positive control and 1*NTC. Triplicate is recommended for every sample.

8. PCR plate diagram

9. Place the 96-well plate or PCR strips in the Quantitative real-time PCR Thermocycler. Run the following qPCR program to acquire FAM fluorescent intensity.

*Acquire FAM fluorescence data.

#Alternatively, set as the minimal time allowed by your Quantitative real-time PCR thermocycler.

Data Analysis

1. Once the qPCR program is finished, the Ct values will generally be calculated in the default threshold (example shown in figure 1).

2. Export the Ct values into Microsoft Excel or other data analysis software. Determine average Ct Values for each triplicate samples. All Ct values should be below that of the NTC.

3. Generate a standard curve to demonstrate linear correlation between Ct values and the standard titer (log scale), like example shown in figure 2. R² is required to above 0.98, while PCR efficiency 0.90~1.10.

4. Plot the unknown samples Ct values against linear correlation formulation to obtain a equivalent pfu/mL value for each sample.

Figure 1. Amplification plots of the fluorescence intensity against the cycle with horizontal threshold line.

Figure 2. The standard correlation curve between Ct values and the standard titer (log scale), followed calculated data on the right.