Storage and Stability
The kit should be stored at -20°C upon arrival. Avoid repeated freeze-thaw cycles. All reagents are stable for up to 12 months when properly stored at -20°C.
Materials Required, Not Provided
These materials are not included in the kit, but will be necessary to use this assay correctly:
– qPCR thermal cycler
– PCR tubes
– 1X Phosphate Buffered Saline or DMEM
The recommended amount of ROX Reference Stain to be added to the MasterMix may vary depending on the type of qPCR machine:
- Without ROX equipment: Not necessary.
- Low ROX equipment: 1 μL/1.25 mL MasterMix.
- High ROX equipment: 11 μL/1.25 mL MasterMix.
1. Sample Preparation: For high titer purified viral samples, dilute virus samples to 107 IU/mL range with 1X phosphate buffered saline or DMEM. For samples with low viral titers, Collect viral supernatant for direct qPCR.
2. Viral Lysis: Add 2 μL of the sample preparation (from step 1) to 18 μL of Viral Lysis Buffer and incubate at room temperature (RT) for 3 min. Use the lysed sample for the reaction.
ΔNote: The viral sample has been diluted 1/10, so take this dilution factor into consideration when calculating the final title.
3. Standard Control DNA Dilutions: Make five 10-fold serial dilutions of the standard Control DNA by diluting 5 μL of DNA in 45 μL of nuclease-free water at each step. dilutions 1/100 to 1/100,000 will be used to generate the standard curve.
4. Setup: It is recommended to set up all reactions on ice in duplicate.
Plot the Ct value (Y-axis, linear scale) against the virus titer (X-axis, logarithmic scale). generate a logarithmic regression using the four (4) standard control DNA dilutions to determine the unknown virus sample titer using y = mln(x) + b from the trend line equation. The R2 value should be > 0.95 to justify the appropriate assay setup. Note to include the dilution factor at the end calculation (i.e. if you diluted your purified viral sample 1/100 in Step 1, then the titer of the unknown sample must be multiplied by a factor of 100).