Ribo Virus
Contents
1. Buffer RAW 30 ml Buffer RAV3 concentrate 12 5 ml Buffer RE 5 ml Carrier RNA lyophilized 1 mg Ribo Virus columns 50 e Collecting tubes 2ml 200 Contains reagents for 50 tests MATERIALS REQUIRED BUT NOT PROVIDED Biological cabinet Ethanol 96 100 Microcentrifuge tubes 1 5 ml Sterile RNase free pipette tips with aerosol barrier Disposable gloves powderless Microcentrifuge with rotor for 2 ml tubes PRODUCT USE LIMITATIONS Ribo Virus is intended as general purpose device No claim or representation is intended for their use to identify any specific organism or for clinical use diagnostic prognostic therapeutic or blood banking It is rather the responsibility of the user to verify the use of the Ribo Virus kits for a specific application range as the performance characteristic of this kit has not been verified to a specific organism DNA viruses e g HBV are usually more difficult to isolate and require proteinase K digestion can be ordered separately WARNINGS AND PRECAUTIONS A Components RAV1 and RAW contain guanidine thiocyanate Guanidine thiocyanate is harmful if inhaled or comes into contact with skin or if swallowed Contact with acid releases toxic gas Xn R 20 21 22 36 38 S 36 37 39 Risk Phrases R 20 21 22 Harmful by inhalation in contact with the skin and if swallowed R 22 Harmful if swallowed R 36 38 Irritating to eyes and skin Safety Phrases S 13 Keep away from food2. aliquot of elution buffer water 1 Add 600 pl buffer RAV1 containing carrier RNA into a 1 5 ml microcentrifuge tube Add 150 pul plasma serum urine cell culture supernatant or cell free body fluid to the Buffer RAV1 Carrier RNA in the microcentrifuge tube Mix by pulse vortexing for 15 sec Incubate for 5 min at 70 C For the isolation of viral DNA add 20 ul proteinase K 20 mg ml stock solution to the lysis mixture not included in this kit but can be ordered separately Incubation time and temperature are critical for lysis as well as RNA stability see trouble shooting for further hints If the resulting solution is still turbid centrifuge the mixture for 1 min at 11 000 x g to pellet particles to prevent clogging of the columns Take off the supernatant and proceed with step 2 Add 600 pl ethanol 96 100 to the sample and mix by vortexing for 15 sec Place Ribo Virus columns in 2 ml centrifuge tubes and load 700 ul lysed sample Centrifuge for 1 min at 8 000 x g Load the residual lysis solution onto the Ribo Virus column Centrifuge for 1 min at 8 000 x g The use of new 2 ml collecting tubes for every step is recommended if infectious material has to be prepared This avoids cross contamination and contamination of centrifuge units Additional collecting tubes can be ordered separately For non infectious samples we recommend to discard the flow through and reuse the 2 ml tube for loading and washing steps Discard
3. flow through and put the Ribo Virus column into another new 2 ml collecting tube More than two loading steps are not recommended Add 500 ul buffer RAW to the Ribo Virus column Centrifuge for 1 min at 8 000 x g Discard flowthrough Add 600 ul buffer RAV3 to the Ribo Virus column Centrifuge for 1 min at 8 000 x g Discard flowthrough Place the Ribo Virus column in a new 2 ml collecting tube and add 200 pl buffer RAV3 Centrifuge for 5 min at 11 000 x g to remove ethanolic buffer RAV3 completely Incubate the columns with open cap for 1 min at 70 C to remove any remaining traces of ethanol Place the Ribo Virus column into a new sterile 1 5 ml centrifuge tube not provided Add 50 ul Buffer RE preheated to 70 C and incubate for 1 2 min Centrifuge for 1 min at 11 000 x g Viral RNA is stable for up to one year when stored at 20 C or 70 C SHORT PROTOCOL Step Description 600 pl RAV1 150 ul sample 70 C 5 min Load sample 1 min 8000 x g 1 wash 500 pl RAW 2 wash 600 ul RAV3 3 wash 200 ul RAV3 1 and 2 1 min 8000 x g 3 5 min 11000 x g 50 ul Buffer RE 70 C 1 2 min 1 min 11000 x g Sacace Biotechnologies Srl 18 San Carlo str 81100 Caserta Italy
4. spin columns all buffers and reagents can be stored for up to 2 years under the above conditions without showing any reduction in performance Before use add 1 ml lysis buffer RAV1 to the complete contents of the carrier RNA tube Dissolve the RNA and transfer it back to the RAV1 bottle Storage of carrier RNA in buffer RAV1 Buffer RAV1 including carrier RNA can be stored at room temperature for 1 2 weeks Storage at room temperature prevents salt precipitation and avoids prewarming the buffer solution Buffer RAV1 including carrier RNA can be stored at 4 C for up to 4 weeks or aliquoted and stored at 20 C for longer periods Storage at 4 C or below may cause salt precipitation Therefore the mixture must be prewarmed at 40 60 C for a maximum of 5 min in order to redissolve salts Do not warm buffer RAV1 containing carrier RNA more than 4 times Frequent warming temperatures gt 80 C and extended heat incubation will accelerate the degradation of carrier RNA This leads to reduced recovery of viral RNA and eventually false negative RT PCR results in particular if lowtiter samples are used Before starting any Ribo Virus protocol prepare the following Buffer RAV3 Add 50 ml ethanol 96 100 to buffer RAV3 Store buffer RAV3 at room temperature for up to one year PROTOCOL Viral RNA DNA isolation from cell free biological fluids with Ribo Virus Before starting the viral RNA isolation prepare a 70 C incubation block and preheat an
5. _ sacace BIOTECHNOLOGIES un K 2 C 22 01 08 Ribo Virus USER MANUAL NAME Ribo Virus INTENDED USE Kit Ribo Virus is designed for the rapid preparation of highly pure viral nucleic acids e g HCV HIV HAV HDV Enteroviruses CMV HBV from fluid biological samples e g plasma serum urine bone marrow swabs liquor PRINCIPLE OF ASSAY With the Ribo Virus RNA viruses are lysed quickly and efficiently by lysis buffer RAV1 which is a highly concentrated solution of GITC Lysis buffer and ethanol create appropriate conditions for binding of nucleic acids to the silica membrane in the Ribo Virus columns Carrier RNA improves binding and recovery of the low concentrated viral RNA Contaminations potential PCR inhibitors like salts metabolites and soluble macromolecular cellular components are removed in simple washing steps with ethanolic buffers RAW and finally RAV3 The nucleic acids can be eluted in low salt buffer or water and are ready for use in subsequent reactions The prepared nucleic acids are suitable for applications like automated fluorescent DNA sequencing RT PCR or any kind of enzymatic manipulation The detection limit for certain viruses depends on individual detection procedures e g in house nested RT PCR We highly recommend the use of internal standards as well as positive and negative controls in order to monitor the purification amplification and detection processes MATERIALS PROVIDED Buffer RAV1 35 ml
6. drink and animal feedstuffs e Wear disposable gloves laboratory coats and eye protection when handling specimens and reagents Thoroughly wash hands afterward Do not pipette by mouth Do not eat drink smoke apply cosmetics or handle contact lenses in laboratory work areas Do not use a kit after its expiration date Dispose of all specimens and unused reagents in accordance with local regulations Specimens should be considered potentially infectious and handled in biological cabinet in accordance with Biosafety Level 2 or other appropriate biosafety practices e Clean and disinfect all spills of specimens or reagents using a disinfectant such as 0 596 sodium hypochlorite or other suitable disinfectant e Avoid contact of specimens and reagents with the skin eyes and mucous membranes If these solutions come into contact rinse immediately with water and seek medical advice immediately e Material Safety Data Sheets MSDS are available on request e Use of this product should be limited to personnel trained in the techniques of DNA amplification e Workflow in the laboratory must proceed in a uni directional manner beginning in the Extraction Area and moving to the Amplification and Detection Area Do not return samples equipment and reagents in the area where you performed previous step SPECIMEN COLLECTION AND CONSERVATION All kinds of biological fluids or semi fluid samples can be processed e g serum urine or BAL For success
7. ful nucleic acid purification it is important to obtain a homogeneous clear and non viscous sample before loading into the corresponding Ribo Virus columns Therefore check all samples especially old or frozen ones for the presence of precipitates Avoid clearing samples by centrifugation filtration before the RAV1 lysis step because viruses of interest may be associated with particles or aggregates Incubation with buffer RAV1 can be prolonged in order to dissolve and digest residual cell structures precipitates and virus particles After collection and centrifugation plasma untreated or treated with anticoagulants other than heparin or serum can be stored at 2 8 C for up to 6 hours For long term storage freezing at 20 C to 80 C in aliquots is recommended Frozen plasma or serum samples must not be thawed more than once Repeated freezing and thawing leads to denaturation and precipitation of proteins causing reduced viral titers and subsequently reduced yields of the isolated viral RNA Samples containing cells such as cerebrospinal fluid bone marrow urine and most swabs should first be filtered or centrifuged for 10 minutes at 1500 x g and the supernatant used STORAGE CONDITIONS AND PREPARATION OF WORKING SOLUTIONS Ribo Virus columns should be stored dry at room temperature 15 25 C storage at higher temperatures should be avoided All solutions should be stored at room temperature unless otherwise stated Ribo Virus
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