96 Well RNA Isolation Kit
Contents
1. DNA RNA method is ready for applications such as RT PCR qPCR differential display microarrays etc Principle The EZgene RNA Isolation kit combines the reversible binding properties of RNA technology with a specially designed buffer system which can effectively remove DNA before RNA isolation Samples are first lysed and homogenized in a specially designed denaturing buffer LCT which immediately inhibits the activity of RNase and DNase The lysate is then passed through a gDNA Clearance Plate which traps the genomic DNA After adjusting the binding condition the flow through lysate that contains RNA are bound to the RNA plate After three wash steps purified RNA is eluted with RNase free water Storage and Stability All components of the 96 Total RNA Kit plus should be stored at 22 C 25 C Under these conditions RNA has successfully been purified and used for RT PCR after 12 months of storage Under cool ambient conditions precipitation may form in Buffer LY and RB Wash Buffer The crystals may be dissolved by heating the buffer at 37 C Page 2 DNase I Digestion Protocol Optional Since the DNA clearance plate eliminates most of the DNA DNase I digestion is not necessary for most downstream applications DNase I set could be bought from Biomiga Note DNase I is very sensitive and prone to physical denaturing do not vortex the DNase I mixture Mix gently by inverting the tube Prepare the fresh DNase I digestion mixt2. Biomiga Inc 10637 Roselle Street Suite C San Diego CA 92121 Tel 858 597 0602 Fax 858 538 1698 Email info biomiga com Visit our web at www biomiga com and learn more about Biomiga products o Biomica The Inventor of EZgene Plasmid Purification System 96 Well RNA Isolation Kit R6811 Handbook VER 2013 06 For in vitro research use only If crystals form in buffers warm up at 37 C to dissolve before use Limited Use and Warranty Related Products Catalog Product Name Preps Price This product is intended for in vitro research use only Not for use in human R6311 01 Tissue RNA kit 150 00 This product is warranted to perform as described in its labeling and in Biomiga s R6311 02 Tissue RNA kit 250 650 00 literature when used in accordance with instructions No other warranties of any R1011 01 RNASecure Solution 45 00 kind express or implied including without limitation implied warranties of R1011 02 RNASecure Solution 150 00 merchantability or fitness for a particular purpose are provided by Biomiga Tissue RNA midi kit Biomiga s sole obligation and purchaser s exclusive remedy for breach of this RGS EOI TSRM mA O 80 00 warranty shall be at the option of Biomiga to replace the products Biomiga shall R6312 02 Tissue RNA midi kit 160 00 have no liability for any direct indirect consequential or incidental damage arising R6314 01 Tissue RNA maxi kit 120 00 out of the use the results of use or
3. e applications 1 X RNA Wash Buffer must be stored and used at room temperature Repeat wash with RNA Wash Buffer DNA DNA contamination Digest with RNase free DNase I Solution contamination Low Abs RNA diluted in acidic DEPC treated water is acidic and can ratios buffer or water dramatically lower Abs260 values Use TE buffer to dilute RNA prior to spectrophotometric analysis Page 12 Table of Contents THOM UCHON 4 seria nine e bens a hese e ee E EERTE Tllustrated Protocol cccccccccceccscseceesssceececssssssscccessessssscessssssecesseee ees Kit Contents Storage and Stability Preparing Reagents Cleaning Plates cece eeeeeceeeeeteeneneee ees Total RNA purification Spin Protocol e cece eee eee cesses eee eae Total RNA purification Vacuum Spin Protocol eeeeeee eee Optional DNase I Digestion Protocol 00 cece ce eenee eee neeetteeee teens Troubleshooting Guide Page 1 Introduction Genomic DNA contamination is a major challenge in the RNA purification process The most common method to remove genomic DNA contaminates is to use DNase digestion The EZgene 96 HP RNA Isolation Kits are designed for fast isolation of total cellular RNA in high throughput format without DNase I digestion By using a special filter plate genomic DNA can be effectively removed The whole protocol can be completed in less than 40 minutes RNA purified using the EZgene
4. entilate the manifold 11 Remove the RNA plate from the top plate of the vacuum manifold and place the RNA plate on top of a 2 ml deep well plate 12 Centrifuge at 5000 x g for 5 minutes to dry the membrane 13 Remove the RNA plate and place it on top of a new 500 ul collection plate supplied with the kit 14 Add 75 100 uL of DEPC treated water to each well Seal the plate with a sealing film Make sure to add water directly onto the center of RNA matrix Incubate for 3 minutes at room temperature Centrifuge at 5 000 x g for 5 minutes at room temperature to elute the RNA 15 Reload the eluted RNA back to the RNA plate for a 2 elution yields another 20 30 of the RNA The first elution normally yields 60 70 of the RNA Page 10 96 Well RNA Isolation Flowchart 4 AAAA Lyse Samples Adjust binding condition and load samples to RNA plate Wash 3x Elute Page 3 Kit contents Catalog R6818 00 R6818 01 R6818 02 96 RNA Plate 1 4 12 96 DNA Clearance Plate 1 4 12 2 mL Deep Well Plate 2 4 24 500 uL 96 Well Plate 1 4 12 Buffer LY 20 mL 125 mL 400 mL RB Wash Buffer 70 mL 280 mL 850 mL RNA Wash Buffer 40 mL 160 mL 2 x 200 mL DEPC Water 10 mL 40 mL 120 mL User Manual 1 1 1 96 Well Collection Plates 2 ml are reusable see Page 5 for instructions Buffer LY and RB Wash Buffer contains a chaotropic salt Use gloves and protective eye wear when handling thi
5. epare Buffer LY and RNA Wash Buffer According to Preparing Reagents Section Procedure 1 A LYSIS OF MONOLAYER CULTURED CELLS GROWN IN A MULTI WELL TISSUE Remove the medium in culture plate by pipetting Add 150 uL of Buffer LY directly to each well Mix thoroughly by pipetting up and down 10 times B LYSIS OF SUSPENSION CULTURED CELLS Transfer aliquots of up to 5 x 10 cells into the wells of a 96 well microplate Spin the plate at 300 x g for 5 minutes Remove the medium completely by pipetting Add 150 uL of Buffer Buffer LY directly to each sample Mix thoroughly by pipetting up and down 10 times Note Add 20 ul 8 mercaptoethanol per 1 ml of Buffer LY before use The complete removal of supernatant is critical for the RNA isolation Page 8 Spin Protocol Materials to be provided by user e 96 100 ethanol e 70 ethanol e Multichannel pipette e RNase free filter pipette tips e Reagent reservoirs for multichannel pipettes e Centrifuge with rotor for 96 well plates e Disposable latex gloves e 2ml96 well deep well plate e Sealing film e Swing Bucket Centrifuge capable of 5 000 x g and Adaptor for 96 Deep Well plates Before Starting e Prepare Buffer LY and RNA Wash Buffer According to Preparing Reagents Section Procedure 1 A LYSIS OF MONOLAYER CULTURED CELLS GROWN IN A MULTI WELL TISSUE Remove the medium in the culture plate by pipetting completely Add 150 uL of Buffer LY directly to each well Mix th
6. o the each well of the RNA plate and centrifuge at 5 000 x g for 5 minutes at room temperature Discard the flow through and reuse the 2 mL Deep Well Plate 7 Add 600 ul of RNA Wash Buffer to each well of the RNA plate Centrifuge at 5 000 x g for 5 minutes at room temperature Discard the flow through and re use the 2 ml deep well Plate 8 Add 600 ul of RNA Wash Buffer to each well of the 96 RNA plate Centrifuge at 5 000 x g for 10 15 minutes at room temperature The prolonged centrifugation is necessary to dry the RNA plate Note It is very important to dry the RNA plate completely before the elution step to remove residual ethanol that might otherwise interfere with downstream applications Page 6 9 Place the RNA plate on top of a 500 ul Elution Plate supplied 10 Add 75 100 ul of DEPC treated water to each well Seal the plate with sealing film Make sure to add water directly onto the center of RNA matrix Incubate for 1 min at room temperature Centrifuge at 5 000 x g for 5 minutes at room temperature to elute the RNA Store RNA at 20 C Note Elution volume can vary according to user preference Page 7
7. oroughly by pipetting up and down 10 times Transfer the cell lysate into a new 2 ml deep well plate supplied B LYSIS OF SUSPENSION CULTURED CELLS Spin down up to 5x 10 5 cells per well at 300 x g for 2 minutes at 4 20 C Remove the medium completely by pipetting Add 150 uL of Buffer LY directly to each well Mix thoroughly by pipetting up and down 10 times Transfer the cell lysate into a new 2 ml deep well plate supplied Page 5 Note Add 20 ul B mercaptoethanol per 1 ml of Buffer LY before use The complete removal of supernatant is critical for the RNA isolation 2 Seal the plate with a sealing film and shake vigorously for 1 min Spin down briefly to avoid cross contamination 3 Remove the sealing film and add 1 volume of 150 uL of 70 ethanol to the sample Seal the plate with the sealing film and mix thoroughly for 1 min by shaking Spin down briefly to avoid cross contamination 4 Place the 96 well RNA plate on top of a 2 ml deep well plate and carefully transfer the entire sample from Step 3 including any precipitate to each well of the RNA plate Note Pipet up and down for 5 times and then transfer to the sample ethanol mix to the 96 well RNA plate 5 Load the RNA plate 2 ml deep well plate into a microplate holder and place the whole assembly into the rotor bucket of the centrifuge Spin at 5 000 x g for 5 minutes at room temperature Discard the flow through 6 Add 500 ul of RB Wash Buffer directly int
8. s solution Preparing Reagents Dilute RNA Wash Buffer with absolute ethanol 96 100 as follows Kit Ethanol to be added R6818 00 Add 160 mL absolute ethanol R6818 01 Add 800 mL absolute ethanol R68 18 02 Add 800 mL absolute ethanol per bottle Buffer LY Add 20 ul 8 mercaptoethanol per 1mL of Buffer LY before use Cleaning of 96 Well collection plates If extra plates are needed please call our customer service department for ordering information To re use the 96 Well collection plates rinse them thoroughly with tap water incubate overnight in 0 2M NaOH 1mM EDTA rinse with distilled water and dry by air Page 4 2 Add 1 volume of 100 ethanol to each well Seal the plate with a sealing film and mix well by shaking for 1 min 3 PREPARE THE VACUUM MANIFOLD Place a 2 ml deep well plate or waste collection tray inside the vacuum manifold base Place manifold s top section squarely over its base Place the RNA plate on the manifold s top section making sure the RNA plate is seated tightly on the rubber ring Connect the vacuum manifold to the vacuum source Keep the vacuum switch off 4 Carefully transfer the entire sample from Step 2 to each well of RNA plate Seal the plate with a sealing film and switch on the vacuum source Apply vacuum until all the sample contents pass through the well membranes Ventilate and turn off the vacuum Note If some of the well is clogged remove the plate and place i
9. t on top of a2 ml deep well plate Centrifuge at 5000 x g for 5 minutes 5 Remove the sealing film and add 500 ul of Buffer RB to each well seal the RNA plate with the sealing film and switch on the vacuum source Apply vacuum until the entire liquid pass through the well membranes Ventilate and turn off the vacuum 6 Add 500 uL of RNA Wash Buffer directly into each well of the RNA plate Apply vacuum until all the liquid passes through the membranes Switch off the vacuum and ventilate the manifold Repeat this step once 7 Remove the RNA plate from the manifold and strike the bottom of the plate on a stack of paper towels Repeat several times until there s no liquid released onto the paper towel 8 Place the RNA plate on top of a 500 uL elution plate Supplied and add 75 100 uL DEPC treated water to each well of the RNA plate Incubate at room temperature for 1 minute 9 Spin at 5 000 x g for 5 min to elute RNA Store RNA at 20 C Page 9 Vacuum Spin Protocol Note that all centrifugation steps must be carried out at room temperature Materials to be provided by user e 96 100 ethanol e Multichannel pipette e RNase free filter pipette tips e Reagent reservoirs for multichannel pipettes e Centrifuge with rotor for 96 well plates e 2ml 96 well deep well plate e Sealing films e Swing Bucket Centrifuge capable of 5 000 x g and Adaptor for 96 Deep Well plates e 96 well Vacuum Manifold Before Starting Pr
10. the inability to use it product R6314 02 Tissue RNA taxi kit 270 00 R6811 01 96 well tissue RNA kit 780 00 R681 1 02 96 well tissue RNA kit 3300 00 R6411 01 Blood RNA mini kit 150 00 R641 1 02 Blood RNA mini kit 680 00 R6812 01 96 well blood RNA kit 780 00 R6812 02 96 well blood RNA kit 20x96 3500 00 For technology support or learn more product information please visit our website at www biomiga com or contact us at 858 597 0602 Page 14 Page 13 Troubleshooting Guide Please use this guide to troubleshoot any problems that may arise For further assistance please contact us at 858 597 0602 Possible Problems and Suggestions Problem Cause Solution Repeat elution step SRS BS RN Aiea onthe Membrane Pre heat DEPC water to 70 C RNA column Membrane is Reduce quantity of starting material overloaded Clogged Incomplete Completely homogenize sample Membrane homogenization Increase centrifugation time Reduce amount of starting material Starting Tissue Freeze starting material quickly in liquid Problems nitrogen Do notstore tissue prior to extraction un less they are lysed first Degraded Pe RNA RNase contamination Follow protocol closely and work quickly Ensure not to introduce RNase during the procedure Check buffers for RNase contamination Problem in Salt carry over Ensure RNA Wash Buffer add ethanol as Downstream during elution indicated on bottl
11. ure before beginning the RNA isolation procedure e Standard DNase buffers may not be compatible with Biomiga s DNase I Digestion Set 1 Follow the standard protocol until the samples completely pass through the RNA Plate Steps 1 6 Then complete the procedure using the following steps A Add 300 RB Wash Buffer to each well of the RNA Plate and centrifuge at 4 000 x g for 1 min B For each RNA sample prepare the DNase I digestion mixture as follows Buffer Volume per Prep DNase I Digestion Buffer 73 5 uL RNase Free DNase I 1 5 uL 20 Kunitz ul Total Volume 75 uL C Pipet 75 uL DNase I digestion mixture directly onto the surface of the membrane in each well of the RNA Plate Be certain to pipet the mixture directly onto each membrane as DNA digestion might not be complete if some of the mixture is retained on the walls or the O rings of the RNA Plate D Incubate at room temperature 15 30 C for 15 minutes 2 Continued proceed to Step 7 on page 6 Spin version or Step 9 on Page 11 9 Add 800 pI RNA Wash Buffer to each well of the RNA plate and apply vacuum until all the liquid passes through the well membranes Switch off the vacuum Note RNA Wash Buffer must be diluted with absolute ethanol before use Refer to label on bottle for directions 10 Add 800 pl of RNA Wash Buffer to each well of the of RNA plate and apply the vacuum until transfer is complete Switch off the vacuum and v
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