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Baculovirus Quantitative Titration Kit STAR

杆状病毒qPCR定量滴定试剂盒

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价格￥2,800.00 （供应商现货： 3-5个工作日）

货号 UA070018

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100Tests

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杆状病毒qPCR定量滴定试剂盒
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背景介绍

The Baculovirus Quantitative Titration Kit serves as an extraction and titration system for fresh (less than 3 months old) budded baculovirus stocks based on AcMNPV baculoviral vectors. Ultra-sensitive TaqMan qPCR technique and accurate titrated virus stocks are employed in this kit, providing an alternatively time-saving method of titration up to 2~3 hours. Therefore, reliable multiplicity of infection (MOI) can be determined real-timely, substituting traditional plaque assays or antibody-based assays which usually take 3~4 days.

The primers and probe included in this kit are specific to gp64 that encodes the essential envelope protein GP64, and the sequences, concentration and ration are also optimized. Virus Lysis Buffer from this kit can degrade viral protein, releasing viral DNA that can be used directly as template for qPCR without further purification. Virus Standard, Positive Control (PC) and negative template control (NTC) should be set up every time of quantitative titration.

产品组分

Components	Amount	Storage
Virus Standard	AcMNPV Virus, 2*10⁸ pfu/mL	-20℃
Virus Lysis Buffer	1mL	-20℃
Proteinase K*	10x, 100 μL	-20℃
Primer & Probe Mix	Forward Primer & Reverse Primer & Probe, 240μL	-20℃ IN THE DARK
qPCR Low ROX mix^†	1.2mL	-20℃
DNA Positive Control	20ng/μL, 25 μL	-20℃
RNase-free Water	1.5mL*3	-20℃

* 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.

操作步骤

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.

	Temperature	Time
Step 1	55℃	30min
Step 2	95℃	5min
Step 3	55℃	4min
Step 4	95℃	1min
Step 5	55℃	1min
Step 6	95℃	30s
Step 7	20℃	Hold

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.

Component	20 µL
qPCR mix（2X）	10µL
Primer&Probe Mix	2µL
Template	2 µL
RNase-free water	To 20 µL

8. PCR plate diagram

2*10⁸pfu/mL	2*10⁸pfu/mL	2*10⁸pfu/mL	Sample1	Sample1	Sample1
2*10⁷pfu/mL	2*10⁷pfu/mL	2*10⁷pfu/mL	Sample2	Sample2	Sample2
2*10⁶pfu/mL	2*10⁶pfu/mL	2*10⁶pfu/mL	Sample3	Sample3	Sample3
2*10⁵pfu/mL	2*10⁵pfu/mL	2*10⁵pfu/mL	Sample4	Sample4	Sample4
2*10⁴pfu/mL	2*10⁴pfu/mL	2*10⁴pfu/mL	Sample5	Sample5	Sample5
2*10³pfu/mL	2*10³pfu/mL	2*10³pfu/mL	Sample6	Sample6	Sample6
PC	PC	PC	…	…	…
NTC	NTC	NTC	…	…	…

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.

Temperature	Time	Cycle
95℃	2min	1x
95℃	5s	40x
60℃*	5s^#	40x

*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.