Contents Introduction
Contents
1. Viral spin columns to which nucleic acid binds while cellular debris hemoglobin and other proteins are effectively washed away High quality nucleic acid is finally eluted in sterile deionized water or low salt buffer Storage and Stability All components of the E Z N A Viral DNA RNA Kit should be stored at 22 C 25 C Under these conditions DNA has successfully been purified and used for PCR after 24 months of storage Under cool ambient conditions a precipitate may form in the Buffer MSL In case of such an event heat the bottle at 37 C to dissolve Store Buffer MSL at room temperature Expiration Date All E Z N A Viral DNA RNA Kit components are guaranteed for at least 24 months from the date of purchase when stored at 22 25 C Binding Capacity Each HiBind Viral column can bind approximately 30 pg nucleic acid Page 2 of 8 Kit Contents Product No R6974 00 R6974 01 R6974 02 Purification times 5 Preps 50 Preps 200 Preps HiBind MicroElute column 5 50 200 2 ml Collection Tubes 15 150 600 Buffer TNA 5 ml 20 ml 60 ml Buffer VHB 4ml 20 ml 60 ml Carrier RNA 150 ug 1 mg 1 mg RWB Wash Buffer 2 ml 12 ml 4x12 ml DEPC Treated Water 2 ml 10 ml 10 ml OB Protease 150 ul 1 5 ml 6 ml User Manual 1 1 1 Before Starting RWB Wash Buffer must be diluted with absolute ethanol 96 100 as follows 1 R6974 00 Add 8 ml absolute ethanol R6974 01 02 Add 48 ml absolute ethano2. HiBind MicroElute Viral column in a 2 ml collection tube provided Transfer the lysate from step 5 into the column and centrifuge at 8 000 x g for 1 min to bind nucleic acid Discard the collection tube and flow through liquid 7 Place the column into a second 2 ml tube provided and wash by pipetting 500 ul of Buffer VHB Centrifuge at 8 000 x g for 1 min Again Discard flow through liquid and reuse the collection tube for next step 8 Place the column into a same 2 ml tube from step 7 and wash by pipetting 500 pl of RWB Wash Buffer diluted with ethanol Centrifuge at 8 000 x g for 1 min Again dispose of collection tube and flow through liquid Note that RWB Wash Buffer is provided as a concentrate and must be diluted Page 4 of 8 10 11 12 with absolute ethanol as indicated on the bottle or page 3 If refrigerated the diluted wash buffer must be brought to room temperature before use Using a new collection tube wash the column with a second 400 ul of RWB Wash Buffer and centrifuge as above Discard flow through and re use the collection tube for next step Place the empty column into the same 2 ml collection tube form step 9 centrifuge at maximum speed 15 000 x g for 2 min to dry the column This step is crucial for ensuring optimal elution in the following step Place the column into a sterile 1 5 ml microfuge tube and add 15 50 ul of DEPC Treated water Allow tubes to sit for 5 min at room temperature T
3. Contents INtrOdUCTION s ea e ess God ee es ROS HO Fee Ow ee Storage and Stability 2 ee ee eens Binding Capacity cs 2 05 da ceeds Saw Gea eye eee ea ee ae a Kit Contents cc ra wk eS ee a ee eS A wee A Before Starting ee sote ee ee ee te tee eee ees A Viral Nucleic acid Spin Protocol 2 ee eee ee eee eee B Viral Nucleic acid Vacuum Protocol 0 cece eee eee eee Troubleshooting Guide Introduction E Z N A MicroElute Viral DNA RNA Kit provides a rapid and easy method for the isolation of viral nucleic acid from plasma serum and other cell free body fluids Samples can be either fresh or frozen provided that they have not been frozen and thawed more than once The kit allows single or multiple simultaneous processing of samples in under 20 minutes There is no need for phenol chloroform extractions and time consuming steps such as CsCl gradient ultracentrifugation and precipitation with isopropanol or ethanol are eliminated DNA purified using the E Z N A MicroElute Viral DNA RNA method is ready for applications such as PCR viral detection and genotyping E Z N A MicroElute Viral DNA RNA Kit uses the reversible nucleic acid binding properties of HiBind matrix combined with the speed of mini column spin technology A specifically formulated buffer system allows viral nucleic acid bind to the matrix Samples are first lysed under denaturing conditions and then applied to the HiBind
4. dure this time making sere to vortex the sample with Buffer MSL immediately and completely Mix thoroughly with Buffer MSL prior to loading HiBind column Page 7 of 8 Problem No nucleic acid eluted Washing leaves colored residue in column Eluted material has red brown color Page 8 of 8 Possible Cause Absolute ethanol not added to Buffer MSL No ethanol added to Wash Buffer Concentrate Incomplete lysis due to improper mixing with Buffer MSL No ethanol added to Wash Buffer Concentrate volume Sample too large Suggestions Before applying sample to column an aliquot of Buffer MSL ethanol must be added See protocol above Dilute Wash Buffer with the indicated volume of absolute ethanol before use Buffer MSL is viscous and the sample must be mixed thoroughly Dilute Wash Buffer with the indicated volume of absolute ethanol before use Reduce sample volume and follow directions
5. l per bottle Carrier RNA must be dissolved with DEPC Water before use and aliquot into adequate portions Store at 20 C IMPORTANT f R6974 00 Add 150ul DEPC water R6974 01 02 Add 1 ml DEPC water Buffer VHB must be diluted with absolute ethanol R6974 00 Add 6 ml absolute ethanol R6974 01 Add 30 ml absolute ethanol R6974 02 Add 90 ml absolute ethanol Page 3 of 8 A Spin Protocol Purification of Viral Nucleic acid from Plasma Serum or whole blood Materials and equipments Supplied by User Tabletop microcentrifuge and sterile 1 5 ml tubes Water bath set to 37 C Ethanol approximately 0 3 ml per sample NOTE The procedure below has been optimized for use with FRESH or FROZEN Plasma Serum or blood samples from 1 to 200ul in volume Other Cell free samples can also be used Bring samples and OB Protease solution to room temperature and have a water bath equilibrated to 37 C Carry out all centrifugation steps at room temperature 1 Transfer 25 pl OB Protease into a sterile microcentrifuge tube 2 Add 200 ul Plasmid or Serum into the tube and bring the volume up to 225 pl with DEPC treated water 3 Add 225 ul Buffer TNA and 4 ul Carrier RNA Mix thoroughly by vortexing 4 Incubate sample at 45 C for 10 min Briefly vortex the tube once during incubation 5 Add 280 ul of isopropanol and mix thoroughly by vortexing Incubate at room temperature for 5 minutes 6 Assemble an
6. ld Insert the column into a collection tube supplied and centrifuge at 15 000 x g for 2 minute to completely dry the column Elute Nucleic acid as Step 11 12 on page 5 Page 6 of 8 Troubleshooting Guide Problem LOW A 60 A280 ratio No Nucleic acid eluted Possible Cause Incomplete lysis Sample too large Sample too viscous Clogged column Poor elution Improper washing Extended centrifugation during elution step Poor cell lysis due t o incomplete mixing with Buffer MSL Poor cell lysis due to improper mixing with Buffer MSL Suggestions Add the correct volume of Buffer MSL and incubate for specified time at 37 C It may be necessary to extend incubation time by 20 min If using more than 200 ul of samples increase volumes of OB Buffer MSL and absolute ethanol Pass aliquots of lysate through one column successively Divide sample into multiple tubes adjust volume to 200 ul with DEPC Treated water See above Repeat elution or increase elution volume Incubation of column at room temperature for 5 min with DEPC Treated water may increase yields Wash Buffer Concentrate must be diluted with absolute 100 ethanol as specified on page 5 before use Resin from the column may be present in eluate Avoid centrifugation at speeds higher than specified The material can be removed from the eluate by centrifugation it will not interfere with PCR or RT PCR Repeat the proce
7. o elute nucleic acid from the column centrifuge at 8 000 x g for 1 min Retain flow through containing the nucleic acid Place column into a second 1 5 ml tube Page 5 of 8 B Vacuum Protocol Purification of Viral Nucleic acid from Plasma or Serum Material and equipments supplied by user Tabletop microcentrifuge and sterile 1 5 ml tubes Vacuum Manifold Water bath set to 37 C Ethanol approximately 0 3 ml per sample Prepare the lysate by following step 1 5 of Protocol A Spin protocol on page 4 Insert the HiBind MicroElute Viral Column into the vacuum manifold Carefully apply the lysate to HiBind MicroElute Viral column Turn on the vacuum source to draw all liquid through the column Turn off the vacuum Note If the lysate has difficulty to pass through the column at this stage Place the column into a collection tube supplied Close the lid and centrifuge at 8 000 x g for 5 minutes or until all liquid pass through the column Place the column into another collection tube supplied and continue step 7 of the spin protocol Pipet 500 ul of Buffer VHB into the column Turn on the vacuum source to draw all liquid through the column Turn off the vacuum Wash the column by pipetting 500 pl of RWB Wash Buffer diluted with ethanol into the column Turn on the vacuum source to draw all liquid through the column Turn off the vacuum Close the lid of HiBind MicroElute Viral column remove it from the vacuum manifo
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