AxyPrep 96-Well Body Fluid Viral DNA/RNA Kit

Transcription

1 AxyPrep 96-Well Body Fluid Viral DNA/RNA Kit Kit contents, storage and stability Cat. No. AP-96-BF- AP-96-BF- AP-96-BF-VNA- VNA-1 VNA-4 12 Kit size well 1.6 ml growblock 96-well DNA plate well V-bottom sample plate silicone mat Adhesive film Proteinase K 33 mg mg mg PK Buffer 2.2 ml 8.5 ml 25.5 ml Buffer AVL 22 ml 85 ml 255 ml Buffer W1A 22 ml 144 ml 192 ml*2 Buffer W2 54 ml*3-39 ml concentrate 10 Buffer W ml concentrate Buffer W2 bottle 1 (empty) Buffer TE (DNase& 7 ml 28 ml 75 ml RNase-free) Protocol Manual Except for Proteinase K, all buffers are stable for a period of at least 12 months when stored under ambient conditions. Please avoid exposure to direct sunlight and extremes in temperature. After reconstitution, Proteinase K is stable for 2 months when stored at 4 C. To preserve Proteinase K activity, the lyophilized Proteinase K is resuspended in PK Buffer, which containing a high concentration of ammonium sulfate. On occasion, a precipitate may form. If this occurs, resuspend it by vortexing or pipetting before use. The Proteinase K activity is unaffected. Proteinase K: Lyophilized Proteinase K is stable for up to 6 months after delivery when stored at room temperature. To prolong the lifetime of Proteinase K, storage at 4 C is recommended. After reconstitution, the Proteinase K is stable for 2 months when stored at 2~8 C. Storing the Proteinase K stock solution at room temperature for prolonged periods of time should be avoided. PK Buffer: Used to dissolve Proteinase K. Store at room temperature. Buffer AVL: Viral lysis buffer. Store at room temperature. Buffer W1A: Wash buffer. Store at room temperature. Buffer W2 concentrate: Desalting buffer. Before use, add the amount of ethanol specified on the bottle label to the W2 concentrate. Either 100% or 95% (denatured) ethanol can be used. page 1

2 Buffer TE(DNase & RNase-free): 5 mm Tris-HCl, ph 8.5, 0.1 mm EDTA, DNase- and RNase-free. Store at room temperature. Introduction Viruses can not only cause serious health troubles but also play an important role in molecular biology and biomedical research. Efficient isolation of viral DNA and viral RNA with high purity and integrity is often a challenge. The AxyPrep 96-well body fluid Viral DNA/RNA Kit provides a rapid method for the purification of viral nucleic acid from 200 μl of body fluid, including plasma, serum, ascites, cell culture supernatant, cerebrospinal fluid, urine, etc. Buffer AVL efficiently lyse the viral particles present in body fluids. During lysis, viral DNA and viral RNA are released and the infective nature of the virus is eliminated. Viral nucleic acid remains soluble in the liquid and is purified by binding to a special 96-well DNA column. After briefly washing with Buffer W1A and Buffer W2 to remove residual impurities and salt, the purified viral nucleic acid is then eluted in Buffer TE (DNase & RNase-free) and can be used immediately. The nucleic acid purified by this method is free from contaminants, such as proteins, pigments, lipids and quantitative PCR/RT- PCR inhibitors, and it is especially suitable for demanding PCR/RT-PCR analyses. Caution 1. Before proceeding with this procedure, make all required preparations to avoid infection by body fluid-borne viral agents. Please follow local guidelines for working with body fluids and infectious agents. 2. Strictly follow all steps in the protocol, and put all waste in an appropriate Biohazardous Waste container Autoclave. 3. Buffer AVL and Buffer W1A contain chemical irritants. When working with the buffers, always wear suitable protective clothing such as safety glasses, laboratory coat and gloves. Be careful to avoid contact with eyes and skin. In the case of such contact, wash immediately with water and seek medical assistance if necessary. Equipment and consumables required Adjustable temperature water bath Centrifuge with swinging bucket rotor and plate carriers (6,000 rpm required) 96 Round-well block (not provide) % ethanol Isopropanol HAc Preparation before experiment 1) Adjust water bath to 56 C. 2) Before the use of the kit, add ethanol to the Buffer W1A and Buffer W2 concentrate as much as specified on the bottle labels. Mix well. 3) Resuspend Proteinase K in PK Buffer. 4) Isopropanol (including 1% HAc) page 2

3 5) Preparation of Buffer W2 (12 96 kit). Add the following to the empty bottle labeled Buffer W2 Bottle provided in the kit: 48 ml 10 Buffer W2 concentrate 432 ml deionized H 2 O 1120 ml ethanol (100% or 95% denatured) Protocols 1. Pipette 20 μl of Proteinase K solution into each well of the Round-well block(not provide). 2. Add 200 μl of a body fluid sample to each well of the Round-well block, Deposit the samples into the wells without wetting the rims Note: If the body fluid sample volume is less than 200 μl, supplement with PBS. 3. Add 200 µl of Buffer AVL to each well of the Round-well block, taking care not to wet the rims of the wells. Seal the wells using a Round-well silicone mat. Note: Be sure that the wells are properly sealed to avoid leaks during shaking. 4. Mix thoroughly by shaking vigorously for 30 seconds. IMPORTANT: For efficient lysis, it is essential that the samples and Buffer AVL are mixed immediately and thoroughly to yield a homogeneous solution. Hold the sealed Round-well Block with both hands and shake up and down vigorously. Inversion or vortexing will not provide sufficient force. 5. Centrifuge briefly at 3,000 rpm to collect any solution from the Round-well silicone mat. Allow the Note: Be sure to use a counterbalance when processing samples in a single Round-well Block. 6. Incubate at 56 C for at least 10 minutes in an incubator or oven. Note: Longer incubation time has no effect on the quality of the purified DNA/RNA. 7. Centrifuge briefly at 3,000 rpm to collect any lysate from the Round-well silicone mat. Allow 8. Remove the Round-well silicone mat and add 250 µl of isopropanol (including 1%HAc) to each well. 9. Seal the wells using the Round-well silicone mat. Shake vigorously for 15 seconds. 10. Centrifuge briefly at 3,000 rpm to collect any solution from the Round-well silicone mat. Allow the 11. Place a 96-well DNA plate onto a 1.6 ml growblock. Remove the Round-well silicone mat from the Round-well block. Transfer the clarified liquid from step 10 to the wells of the DNA plate. Seal the top of the 96-well DNA plate with a piece of clear BF-400 breathable film. Centrifuge at 6,000 rpm for 1 minute. 12. Discard the filtrate from the wells of the 1.6 ml growblock. Place the DNA plate back onto the 1.6 ml growblock. Add 500 μl of Buffer W1A to the 96-well DNA plate. Seal the top of the 96-well DNA plate with a piece of clear BF-400 breathable film. Centrifuge at 6,000 rpm for 1 minute. Note: Make sure that ethanol has been added into Buffer W1A. 13. Discard the filtrate from the wells of the 1.6 ml growblock. Place the DNA plate back onto the 1.6 ml growblock. Add 700 μl of Buffer W2 along the walls of the DNA plate wells to wash off any page 3

4 residual Buffer W1A. Seal the top of the 96-well DNA plate with a piece of clear BF-400 breathable film. Centrifuge at 6,000 rpm for 1 minute. Note: Make sure that ethanol has been added into Buffer W2 concentrate. 14. Discard the filtrate from the wells of the 96-well 1.6 ml growblock. Place the DNA plate onto the 1.6 ml growblock, add 500 μl of Buffer W2 to the DNA plate. Seal the top of the 96-well DNA plate with a piece of clear BF-400 breathable film. Centrifuge at 6,000 rpm for 1 minute. Note: Make sure that ethanol has been added into Buffer W2 concentrate. 15. Discard the filtrate from the wells of the 96-well 1.6 ml growblock. Place the DNA plate onto the 1.6 ml growblock, Seal the top of the 96-well DNA plate with a piece of clear BF-400 breathable film. Centrifuge at 6,000 rpm for 2 minutes. 16. Place the 96-well DNA plate onto a 96-well V-bottom sample plate. To elute the viral DNA/RNA, add 50 μl of Buffer TE (DNase& RNase-free), and let it stand for 2 minutes at room temperature. Seal the top of the DNA plate with a piece of clear BF-400 breathable film. Centrifuge at 6,000 rpm for 2 minutes. Note: To prevent viral RNA degradation, we recommend adding RNasin at 1 unit/μl in TE eluent when viral RNA is purified. Overview Add 20 μl of Proteinase K Add 200 μl of samples Add 200 μl of Buffer AVL Incubate at 56 C for 10 min Lysis Add 250 μl of isopropanol(including 1%HAc) Transfer the lysate to the 96-well DNA plate Add 500 μl of Buffer W1A Add 700 μl of Buffer W2 Repeat wash with 500 μl Buffer W2 Binding Washing Add 50 μl of Buffer TE (DNase & RNasefree) Elution page 4

5 Troubleshooting 1. PCR or RT-PCR generates multiple amplicons or wrong size amplicon This problem is generally attributable to contamination of the plasticware or PCR reagents. If amplicons are present in addition to the correct amplicon, there is most likely a contamination issue. However, a negative controls (absence of viral DNA/RNA template) should be run to verify the source of the contamination. Plasticware and/or reagents should be replaced as necessary. 2. PCR fails to generate amplicons Problem with PCR primers, reagents or cycling parameters Run the appropriate control reactions to verify the integrity of all PCR components. Failure to add ethanol to Buffer W2 concentrate (premature elution of bound viral DNA) Inspect the volume of Buffer W2 in the bottle or review any notes made on the label to confirm the addition of ethanol. Inspect ethanol source to confirm that it was 95% (denatured) or 100% ethanol NOT 70% ethanol. If Buffer W2 is suspect, the procedure can be repeated, substituting 70% ethanol for Buffer W2. Low viral titer in sample Increase the amount of viral DNA to the maximum extent possible and repeat the PCR. Increase the number of cycles in the PCR. Set up a control PCR in which the viral DNA is replaced with another template. Degradation of viral DNA The limited amounts of viral nucleic acid present in most biological fluid samples demands that care and cleanliness be exercised when purifying. If necessary, repeat the procedure after carrying out the appropriate measures to remove any potential sources of contamination, etc. 3. RT-PCR fails to generate amplicons Problem with RT-PCR primers, reagents or cycling parameter Run the appropriate control reactions to verify the integrity of all RT-PCR components. Low viral titer in sample Increase the amount of viral RNA to the maximum extent possible and repeat the RT-PCR. Increase the number of cycles in the RT-PCR. Set up a control RT-PCR in which the viral DNA is replaced with another template. Degradation of viral RNA Given the limited amount of viral RNA which is likely to be recovered from biologic fluid samples, it is particularly important that great care and cleanliness be excercised when preparing, storing and handling these samples. If necessary, repeat the procedure after carrying out the appropriate measures to remove any potential sources of contamination, etc. Use DEPC-treated materials whenever practical. Add RNasin (1 unit/μl) to the purified viral RNA for stability during storage. page 5