Viral RNA+DNA Preparation Kit

Spin column based RNA+DNA purification from blood, serum, tissue or cell culture

Description:
Viral RNA+DNA Preparation Kit is designed for rapid and effective isolation of RNA and DNA from a variety of pathogen organisms such as virus or bacteria. Samples can be fresh or frozen plasma/blood (treated with anticoagulants except heparin), serum, other cell-free body fluids or pathogen-infected tissue. The kit allows high yield isolation of viral RNA/DNA from nasal or throat swabs.
The kit is specifically designed to isolate high-quality nucleic acids using low elution volumes and allowing sensitive downstream analysis including quantitative PCR and RT-PCR. The purified RNA/DNA is free of proteins and nucleases. Viral RNA+DNA Preparation Kit uses lysis buffer including chaotropic salts to inactivate RNases/DNases and advanced silica-gel membrane technology for fast purification of intact RNA/DNA. The preparation procedure is optimized to achieve reliable results within 30 min.

Preparation procedure:
The DNA purification follows a cell lysis, RNA/DNA binding, washing and eluting procedure. Before starting, add Ethanol (96-99 %, not included in the kit) to Washing Buffer A and B as indicated on data sheet and bottles. Please note that the Ethanol concentration of a Washing Buffer may decrease during long term storage resulting in a drop-down of the final DNA yield.
The provided Lysis Buffer contains carrier molecules to enhance binding of RNA/DNA on the column membrane.

1a Preparation from nasal or throat swabs

• Transfer 250 μl of Lysis Buffer into a microcentrifuge tube.
• Cut off the cotton tip with the collected nasal or throat cells and place it in the micro tube.
• Close the tube and vortex for 15 sec.
• Incubate at room temperature (20-25 °C) for 10 min.
• Remove the cotton tip and squeeze it out at the rim of the tube.
• Add 250 μl Ethanol (96-99 %) and mix well by gently vortexing.

1b Preparation from plasma, serum, urine, cell-culture supernantant, cell-free fluid or virus infected tissue

• Transfer 100 μl plasma, serum, urine, cell-culture supernatant, cell-free fluid or virus infected tissue into a 1.5 ml microtube.
• Note: Samples of larger volumes (up to 200 μl) can easily be scaled up but may require larger tubes for the lysis procedure.
• Add 250 μl (or 2.5 amounts of sample volume) of Lysis Buffer.
• Vortex for 15 sec.
• Incubate at room temperature (20-25 °C) for 10 min.
• Add 250 μl (or 2.5 amounts of sample volume) Ethanol (96-99 %) and mix well by gently vortexing.

2 Column Loading

• Place a Spin Column into a provided 2 ml collection tube.
• Spin down the Lysate-Ethanol mixture and transfer the solution into the Spin Column.
• Close the cap and centrifuge the Spin Column at 13,000 rpm for 1 min.
• Discard the flow-through in the collection tube and place the column back in the same tube.
• Note: The maximum volume of the column reservoir is 800 μl. For larger sample volumes discard the flow-through in-between and load the spin column again.

3 Column Washing

• Add 500 μl Washing Buffer A (Ethanol added) to the Spin Column and centrifuge at 13,000 rpm for 1 min.
• Discard the flow-through in the collection tube and place the Spin Column back in the same tube.
• Add 500 μl Washing Buffer B (Ethanol added) to the Spin Column and centrifuge at 13,000 rpm for 1 min.
• Note: Before using Washing Buffer B for the first time add ethanol as indicated on the bottle.
• Discard the flow-through in the collection tube and place the Spin Column back in the same tube.
• Centrifuge at 13,000 rpm for 1 min.
• Note: It is important to dry the membrane since residual ethanol may interfere with downstream reactions.

4 Elution

• Place the Spin Column into a new 1.5 ml microtube (not provided).
• Add 40-50 μl Elution Buffer directly onto the membrane of the spin column.
• Note: Avoid touching membrane with the pipet tip.
• Incubate at room temperature for 1 min.
• Centrifuge at 13,000 rpm for 1 min.
• Use 2-5 μl of the eluted RNA and/or DNA as template in PCR or RT-PCR assays or for further down-stream applications. The eluted RNA/DNA can be stored at -70 °C.