MicroElute Viral RNA/DNA Kit. R preps R preps R preps

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1 MicroElute Viral RNA/DNA Kit R preps R preps R preps July 2012

2 MicroElute Viral RNA/DNA Kit Table of Contents Introduction and Overview...2 Kit Contents/Storage and Stability...3 Preparing Reagents...4 Important Notes...5 MicroElute RNA/DNA Protocol...6 Troubleshooting Guide...8 Manual Revision: July 2012 Innovations in nucleic acid isolation 1

3 Introduction and Overview The MicroElute Viral RNA/DNA Kit is designed for easy and rapid isolation of viral RNA and DNA from whole blood and acellular fluids such as plasma, serum, urine, and cell culture supernatant. The procedure completely removes contaminants and enzyme inhibitors, thereby producing high-quality viral RNA/DNA. RNA and DNA purified using the MicroElute Viral RNA/DNA method is ready for all downstream amplifications applications. The MicroElute Viral RNA/DNA Kits use MicroElute technology to purify DNA/RNA in small elution volume. Up to 200 µl sample is mixed with VRS Buffer and Proteinase K Solution to lyse the viral particles. After adjusting the binding condition with ethanol, the sample is loaded on the MicroElute RNA Mini Column to bind the DNA and RNA. With three quick wash steps, purified viral RNA and DNA is eluted with water. New in this Edition: This manual has been edited for content and redesigned to enhance user readability. Proteinase K is now supplied in a liquid form eliminating the resuspension step prior to use. Proteinase K Solution can be stored at room temperature for 12 months. Proteinase Storage Buffer is no longer included in this kit. 2

4 Kit Contents Product R R R Purifications MicroElute RNA Mini Column ml Collection Tubes VRS Buffer 1.5 ml 15 ml 60 ml RWF Buffer 5 ml 30 ml 120 ml RNA Wash Buffer II 5 ml 12 ml 50 ml Proteinase K Solution 150 µl 1.5 ml 6 ml Carrier RNA (Poly A) 50 µg 500 μg 2 mg DEPC Water 1.5 ml 10 ml 30 ml User Manual P P P Storage and Stability All components in the MicroElute Viral RNA/DNA Kit should be stored at room temperature except for Carrier RNA. Proteinase K Solution can be stored at room temperature for 12 months. For long-term storage (>12 months), store Proteinase K Solution at 2-8 C. Carrier RNA must be stored at 2-8 C. Once reconstituted, store the Carrier RNA at -20 C. During shipping and storage, crystals may form in the VRS Buffer, simply warm to 37 C to dissolve. All kit components are guaranteed for at least 12 months from date of purchase. 3

5 Preparing Reagents 1. Dissolve Carrier RNA in DEPC Water as follows, divide into convenient aliquots, and store at -20 C. Kit DEPC Water to be Added R μl R μl R ml 2. Dilute RNA Wash Buffer II with 100% ethanol as follows and store at room temperature. Kit 100% Ethanol to be Added R ml R ml R ml 4

6 Important Notes 1. Carrier RNA should be dissolved in DEPC Water to obtain a 1 µg/µl solution. Dissolve the Carrier RNA thoroughly, divide it into convenient aliquots, and store at -20 C. DO NOT frequently thaw-freeze the Carrier RNA solution. It is recommended to make aliquots of this buffer according to average usage per week. 2. Please take a few minutes to read this booklet thoroughly and become familiar with the protocol. Prepare all materials required before starting to minimize RNA degradation. 3. Whenever working with RNA, always wear gloves to minimize RNase contamination. Use only clean RNase-free pipette tips when using the supplied reagents. 4. During the procedure work carefully but quickly. 5. Carefully apply the sample or solution to the center of the MicroElute RNA Mini Column. Avoid touching the membrane with the pipette tip. 6. Sample volume: The MicroElute RNA Mini Column can bind RNA greater than 200 nt. Yield will depend on the sample source and conditions. The protocol is optimized for use with 1 ml samples. If the sample volume is less than 1 ml, adjust the volume to 1 ml with PBS buffer. Quantitation and Storage of RNA To determine the concentration and purity of RNA, measure absorbency at 260 nm and 280 nm in a spectrophotometer. 1 OD unit measured at 260 nm corresponds to 40 μl RNA per ml. The ratio of A 260 /A 280 of pure nucleic acids is 2.0, while for pure protein it is approximately 0.6. A ratio of corresponds to % pure nucleic acid. Phenol has an absorbency maximum at 275 nm and can interfere with spectrophotometric analysis of DNA or RNA. However, the MicroElute RNA Isolation technology eliminates the use of phenol and avoids this problem. Store RNA samples at -70 C in water. Under such conditions RNA prepared with the MicroElute system is stable for more than a year. 5

7 MicroElute Viral RNA/DNA Kit Protocol MicroElute Viral RNA/DNA Kit Protocol Materials and Equipment to be Supplied by User: 100% ethanol Sterile RNase-free pipette tips Nuclease-free 1.5 ml and 2 ml microcentrifuge tubes Table top microcentrifuge at room temperature Vortexer Phosphate-buffered saline (PBS) Shaking Incubator or heating block set to 37 C Before Starting: Prepare Carrier RNA and RNA Wash Buffer II as instructed in the Preparing Reagents section on Page 4. Equilibrate samples and all buffers to room temperature before beginning. 1. Add 200 µl sample into a 2 ml microcentrifuge tube. Note: If the sample volume is less than 200 µl, adjust the volume to 200 µl with PBS. 2. Add 210 μl VRS Buffer, 8 µl Carrier RNA, and 20 µl Proteinase K Solution (20 mg/ml). Vortex for 30 seconds to mix thoroughly. 3. Incubate at 37 C for 10 minutes. 4. Centrifuge briefly at maximum speed to collect any liquid drops from the tube cap. 5. Add 220 µl 100% ethanol. Vortex for 20 seconds to mix thoroughly. 6. Let sit at room temperature for 5 minutes. 7. Insert at MicroElute RNA Mini Column into a 2 ml Collection Tube. 6

8 MicroElute Viral RNA/DNA Kit Protocol 8. Transfer the entire sample (~ 650 µl) to the MicroElute RNA Mini Column. Note: The maximum capacity of the MicroElute RNA Mini Column is 800 μl. Larger volumes can be loaded successively. Work quickly, but carefully. 9. Centrifuge at 6,000 x g for 1 minute. 10. Discard the filtrate and the collection tube. 11. Transfer the MicroElute RNA Mini Column to a new 2 ml Collection Tube. 12. Add 500 µl RWF Buffer. 13. Centrifuge at 10,000 x g for 30 seconds. 14. Discard the filtrate and reuse the collection tube. 15. Add 700 µl RNA Wash Buffer II. 16. Centrifuge at 10,000 x g for 30 seconds. 17. Discard the filtrate and reuse the collection tube. 18. Centrifuge the empty MicroElute RNA Mini Column for 1 minute at maximum speed to dry the column matrix. 19. Transfer the MicroElute RNA Mini Column to a clean 1.5 ml microcentrifuge tube. 20. Add μl DEPC Water. Add the water directly onto the center of the column matrix. 21. Centrifuge at maximum speed for 1 minute. 22. Store RNA/DNA at -70 C. 7

9 Troubleshooting Guide Please use this guide to troubleshoot any problems that may arise. For further assistance, please contact the technical support staff, toll free, at Problem Cause Solution Little or no RNA eluted Carrier RNA was not added to VRS Buffer or degraded RNA remains on the column Column is overloaded Ethanol was not added to RNA Wash Buffer II Problem Cause Solution Degraded RNA Problem in downstream applications Source RNase contamination Salt carry-over during elution Repeat with new sample and make sure to add Carrier RNA. Avoid frequent freeze-thaw cycles of the Carrier RNA. Repeat the elution step. Preheat DEPC Water to 70 C prior to elution. Incubate column for 5 minutes with DEPC Water prior to centrifugation. Reduce the volume of starting material Increase the incubation time of Proteinase K digestion Check the bottle label (or Page 4) and add the appropriate amount of ethanol to RNA Wash Buffer II Do not freeze-thaw the sample more than once. Follow the protocol closely and work quickly. Low virus concentration Ensure not to introduce RNase during the procedure. Check buffers for RNase contamination. Ensure RNA Wash Buffer II has been diluted with 4 volumes of 100% ethanol as indicated on bottle. RNA Wash Buffer II must be stored at room temperature. 8