High Pure Viral Nucleic Acid Large Volume Kit
Low to medium throughput viral nucleic acid isolation for large sample volumes.
For general laboratory use.
|Product No.||Pack Size|
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The High Pure Viral Nucleic Acid Large Volume Kit is intended for general laboratory use. The kit efficiently purifies viral nucleic acids from serum, plasma, or whole blood. Based on the same principle as the High Pure Viral Nucleic Acid Kit, the kit features an innovative spin column assembly (see Figure 1) to purify larger sample volumes of up to 2.5 mL. When using whole blood as starting material, total nucleic acids are isolated, including viral nucleic acids. For optimal results, the first step of the isolation process should be performed in a tabletop centrifuge with a swinging-bucket rotor capable of holding 50 mL tubes. The purified viral nucleic acid is eluted in nuclease-free water, and is suitable for direct use in PCR and RT-PCR.
- Improve sensitivity: Use sample volumes up to 2.5 mL to obtain high yields of purified nucleic acid in a concentrated 50 μL eluate.
- Obtain high-purity nucleic acids: Reduce carryover risk by using high centrifugal forces in all wash steps.
- Increase convenience and improve time to result: Eliminate complicated sample pre-processing and rapidly recover purified samples using high-speed centrifugation.
- Binding Buffer
- Poly(A), lyophilized
- Proteinase K, recombinant, lyophilizate
- Inhibitor Removal Buffer
- Wash Buffer
- Elution Buffer
- High Pure Extender Assembly
- Collection Tubes
The High Pure Viral Nucleic Acid Kit Large Volume isolates highly purified viral nucleic acids from up to 2.5 mL mammalian plasma, serum, or whole blood. The purified viral nucleic acid may be used directly in PCR and RT-PCR.
Figure 1: Crossing Points of a series dilution of HAV particles in human citrate plasma after isolation with the High Pure Viral Nucleic Acid Large Volume Kit and subsequent analysis on the LightCycler® 2.0 Instrument.
1 ml citrated plasma was spiked with decreasing amounts of HAV viral particles (1 × 105 to 1 × 102 particles). Isolation was performed according to the Instructions for Use.
Figure 2: Crossing Points of a series dilution of HAV and EBV particles in human serum after isolation with the High Pure Viral Nucleic Acid Large Volume Kit and subsequent analysis on the LightCycler® 2.0 Instrument.
1 ml of negative human serum was spiked with a dilution series (1 × 106 to 1 × 104 particles) of HAV or EBV virus particles, followed by isolation according to the Instructions for Use. Isolation efficiency and quality were analyzed by qPCR and qRT-PCR on the LightCycler® 2.0 Instrument utilizing a quantitative analysis of HAV and LightCycler® EBV Quantification Kit (Roche Molecular Diagnostics, Cat. No. 04 846 818 001), respectively.
Nucleic acids bind to the surface of the glass fiber fleece in the presence of a chaotropic salt (guanidine HCl). This allows the High Pure filter tube to specifically immobilize nucleic acids (both DNA and RNA) while they are freed of contaminants.
Typical Nucleic Acid Recovery
Number of Reactions
|Serum, plasma, whole blood||1 - 2.5 mL||25 minutes||40/2.5 mL|
As a prerequisite for PCR or RT-PCR, viral nucleic acids must be isolated from serum, plasma, or whole blood. Viral lysis is accomplished by incubating the sample in a special Lysis/Binding Buffer in the presence of Proteinase K. After applying the lysis mixture to a High Pure Extender Assembly, centrifugation is performed in a tabletop centrifuge. During the spin, the lysis mixture passes through the glass-fiber fleece of the High Pure Spin Column, and nucleic acids are bound to the silica fibers. After the first centrifugation, the High Pure Spin Column is removed from the Extender Assembly and further processed in a microcentrifuge using 2 mL Collection Tubes. During this process, nucleic acids are purified from salts, proteins, and other impurities by washing and centrifugation steps. The bound nucleic acids are eluted in nuclease-free water and collected after centrifugation.
Figure 1: High Pure Large Volume Assembly.
The innovative, preassembled device features a High Pure Spin Column and extender, facilitating the efficient purifiaction of sample volumes of <2.5 mL without complicated sample pre-processing. High-purity nucleic acids are recovered in 50 μL eluate.
Function-tested with EBV plasmid purified according to the kit protocol. Isolation efficiency is tested in PCR using the LightCycler® Carousel-Based System.