E.Z.N.A. HP Viral RNA/DNA Kit. R preps R preps

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1 E.Z.N.A. HP Viral RNA/DNA Kit R preps R preps August 2013

2 E.Z.N.A. HP Viral RNA/DNA Kit Table of Contents Introduction and Overview...2 Kit Contents/Storage and Stability...3 Preparing Reagents...4 Before Beginning...5 HP Viral RNA/DNA Protocol...6 Troubleshooting Guide...9 Manual Revision: August 2013 Innovations in nucleic acid isolation 1

3 Introduction and Overview E.Z.N.A. HP Viral RNA/DNA Kit is designed for rapid isolation of viral RNA and DNA from 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. By using a procedure that concentrates viral particles before nucleic acid extraction, this kit allows the use of a large starting volume of sample (up to 1 ml) so that the lower titer virus can be detected in the downstream applications. The RNA and DNA purified from using the E.Z.N.A. HP Viral RNA/DNA method is ready for all downstream amplification applications. The E.Z.N.A. HP Viral RNA/DNA Kits use HiBind technology to purify DNA/RNA from larger volume acellular samples. Up to 1 ml of sample is mixed with VRA Buffer to selectively precipitate the viral particles. After the centrifugation, the viral pellet is resuspended with TL Buffer and Proteinase K. After adjusting the binding conditions with VRS Buffer and ethanol, the sample is loaded on a HiBind RNA Mini Column to bind DNA and RNA. After two wash steps, purified viral RNA/DNA is eluted with RNase-free 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 step to resuspend prior to use. Proteinase K Solution can also 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 Purifications 5 50 HiBind RNA Mini Columns ml Collection Tubes VRA Buffer 5 ml 40 ml TL Buffer 1.5 ml 15 ml VRS Buffer 1.5 ml 15 ml RWF Buffer 5 ml 30 ml RNA Wash Buffer II 5 ml 12 ml Proteinase K Solution 150 µl 1.5 ml Carrier RNA (Poly A) 50 µg 500 µg DEPC Water 1.5 ml 10 ml P P Storage and Stability All components in the E.Z.N.A. Viral RNA/DNA Kit should be stored at room temperature except for Carrier RNA. For long-term storage (>12 months), store Proteinase K Solution at 2-8 C. Carrier RNA must be stored at 2-8 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 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 4

6 Before Beginning 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 HiBind RNA Mini Column. Avoid touching the membrane with the pipette tip. 6. Sample volume: The HiBind 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 E.Z.N.A. HP Viral RNA/DNA Kit Protocol E.Z.N.A. HP 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 1 ml sample to a 2 ml microcentrifuge tube. Note: If the sample volume is less than 1 ml, adjust the volume to 1 ml with PBS. 2. Add 720 μl VRA Buffer. Vortex for 30 seconds to mix thoroughly. 3. Centrifuge at 13,000 x g for 5 minutes. 4. Aspirate and discard the supernatant. 5. Add 230 μl TL Buffer and 20 µl Proteinase K Solution (20 mg/ml). Vortex to mix thoroughly. 6. Incubate at 37 C for 10 minutes in a shaking incubator. Note: If a shaking incubator is not available, incubate the sample in a water bath or heat block. Vortex briefly for every 3-4 minutes. 6

8 E.Z.N.A. HP Viral RNA/DNA Kit Protocol 7. Add 240 µl VRS Buffer and 8 µl Carrier RNA. Vortex to mix thoroughly. 8. Incubate at 37 C for 5 minutes. 9. Add 250 µl 100% ethanol into the sample. Vortex for 30 seconds to mix thoroughly. 10. Let sit at room temperature for 5 minutes. 11. Insert a HiBind RNA Mini Column into a 2 ml Collection Tube. 12. Transfer the entire sample (~ 750 µl) to the HiBind RNA Mini Column. Note: The maximum capacity of the HiBind RNA Mini Column is 800 μl. Larger volumes can be loaded successively. Work quickly, but carefully. 13. Centrifuge at 6,000 x g for 1 minute. 14. Discard the filtrate and the collection tube. 15. Insert a HiBind RNA Mini Column into a 2 ml Collection Tube. 16. Add 500 µl RWF Buffer. 17. Centrifuge at 10,000 x g for 30 seconds. 18. Discard the filtrate and reuse the collection tube. 19. Add 700 µl RNA Wash Buffer II. 20. Centrifuge at 10,000 x g for 30 seconds. 21. Discard the filtrate and reuse the collection tube. 7

9 E.Z.N.A. HP Viral RNA/DNA Kit Protocol 22. Centrifuge the empty HiBind RNA Mini Column for 1 minute at maximum speed to dry the column matrix. 23. Transfer the HiBind RNA Mini Column to a clean 1.5 ml microcentrifuge tube. 24. Add μl DEPC Water. Add the water directly onto the center of the column matrix. 25. Centrifuge at maximum speed for 1 minute. 26. Repeat Steps for a second elution step. 27. Store RNA/DNA at -70 C. 8

10 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 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 Ethanol was not added to RNA Wash Buffer II Check the bottle label (or Page 4) and add the appropriate amount of ethanol to RNA Wash Buffer II Problem Cause Solution Degraded RNA Source RNase contamination 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. Problem in downstream applications Salt carry-over during elution 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. 9

11 Notes: 10