Mag-Bind® mRNA Enrichment Kit
Contents
1. become familiar with the procedures The oligo dT magnetic particles are mixed with total RNA solution Poly A RNA hybridizes to the magnetic particles under optimized conditions After applying the magnetic field the magnetic particle mRNA complexes is pulled out of the solution Contaminants are removed by aspiration and then the magnetic beads are thoroughly washed by two quick wash steps Purified mRNA is eluted from the magnetic particles in an aqueous solution New in this Edition The latest manual has been redesigned to enhance readability and layout Binding Capacity 100ul of the Mag Bind oligo dT magnetic beads solution can bind approximately 4 ug of mRNA Kit Contents and Storage Kit Contents Oligo dT Magnetic Beads 110 ul 550 ul 2X Mag Bind mRNA Binding Buffer Mag Bind mRNA Wash Buffer mRNA Elution Buffer Storage and Stability All components of the Mag Bind mRNA Enrichment Kit should be stored at 22 25 C Under these conditions RNA has successfully been purified and used for RT PCR after 12 months of storage Do not freeze the Mag Bind oligo dT magnetic beads solution Working with RNA Please take a few minutes to read this booklet thoroughly to become familiar with the protocol Prepare all materials required before starting to minimize RNA degradation Whenever working with RNA always wear latex gloves to minimize RNase con tamination Change gloves frequently Use only clean RNase free dis2. Ca OMEGA Innovations in nucleic acid isolation bio tek Product Manual Mag Bind mRNA Enrichment Kit R6520 00 2 preps R6520 01 10 preps January 2014 For research use only Not intended for diagnostic testing Mag Bind mRNA Enrichment Kit Table of Contents UCC CU EVO cers fi cecars sents cerew ey nestasnd Amseuere ctcaeeenyaeteatade staker kanki Kit Contents Storage and Stability esscsscsssesssecsecscenseessecneeeses Working with FIN Picea sctaseanasacccaacssetssvacecensaeensscuanacvanactananseeaiactaressteseeatsieess Mag Bind mRNA Enrichment Standard Protocol Mag Bind mRNA Enrichment Centrifugation Protocol Troubleshooting GUIGE siiceicnsisiisinisniiinanioniaineannaamenn Manual Revision January 2014 N OMEGA bio tek Innovations in nucleic acid isolation Introduction High purity mRNA is critical for downstream applications such as RT PCR and QRT PCR The Mag Bind mRNA Purification Kit provides a convenient and rapid method for the isolation of high purity of MRNA from total RNA samples This kit is based on Mag Bind magnetic particles which have a large surface compare to other standard magnetic beads and delivery high purity of mRNA The magnetic bead format also can be easily scaled up and down according to the sample offering scalability and flexibility for a variety of downstream applications If using the Mag Bind mRNA Kit for the first time please read this booklet in its entirety to
3. oid disturbing the magnetic particles pellet 10 11 12 13 14 15 16 17 18 19 Mag Bind mRNA Protocol Centrifugation Protocol Wash the magnetic beads again by adding another 200 ul mRNA Wash Buffer Centrifuge at 8 000 x g for 1 minute to collect the magnetic particles Carefully remove the supernatant with a pipettor Avoid disturbing the magnetic particles pellet Centrifuge at 10 000 x g for 1 minute to collect the magnetic particles Carefully remove the supernatant with a pipettor Avoid disturbing the magnetic particles pellet Dry the magnetic beads pellet by air for 5 10 minutes Remove any liquid with a pipettor Add 100ul of mRNA elution Buffer to the particles Incubate the tube at room temperature with gentle agitation for 5 minutes to release mRNA from the magnetic particles Centrifuge at 10 000 x g for 2 minutes to collect the magnetic beads Transfer the supernatant containing the eluted mRNA into a RNase free tube The RNA can be store at 20 C for short term storage and 80 C for long term storage Optional Precipitate mRNA by ethanol precipitation by adding 10ul of 5 M NaCl and 2 5 volume of cold absolute ethanol Incubate for 20 minutes at 20 C Centrifuge at maximum speed for 10 minutes at room temperature Wash once with 300ul of 70 ethanol and dissolve the purified mRNA with 10 20ul of nuclease free water Troubleshooting Guide Please use this guide to troublesho
4. ot any problems that may arise For further assistance please contact the technical support staff toll free at 800 832 8896 Possible Problems and Suggestions Follow protocol closely and work quickly RNase contamination from handling Wear gloves throughout the proce dure and when handling the solu tion and equipment used for RNA isolation Ensure not to introduce RNase during Degraded RNA the procedure Check Total RNA sample for RNase contamination incubate the total RNA sample at 65C for 5 minutes and from total RNA sample then incubate at room temperature for 10 minutes Analyze the sample by agarose gel electrophoresis RNase contamination can be determined by loss or smear of 18S and 28S rRNA bands Ensure Total RNA sample is heated at 65 C prior to addition of magnetic particles RNase contamination rRNA contamina rRNA co purified with tion mRNA If the rRNA level is too high for down stream application purify the mRNA with second round purification with fresh magnetic particles Completely remove the magnetic particles by magnetic stand or cen trifugation OD260 0D280 Magnetic beads ration is too low interference Optical densities i Make sure to wash Mag Bind pellet Trace contaminants do not agree fied froricolumn as instructed Alternatively rely on with DNA yield agarose gel ethidium bromide elec increase A260 eg on agarose gel trophoresis for quantization pf cau
5. posable plastic pipette tips when using the supplied reagents During the procedure work carefully but quickly Under cool ambient conditions crystals may form in the Mag Bind mRNA Binding Buffer This is normal and the bottle may be warmed to 50 C to redissolve the salt Mag Bind mRNA Protocol Standard Protocol Mag Bind mRNA Protocol Standard Protocol Materials to be provided by user Magnetic Stand for 1 5 ml tube OBI MSD 02 Nuclease free 1 5ml centrifuge tubes Optional Absolute Ethanol chilled at 20 C Incubator capable of 70 C Before Starting Set an Incubator to 65 C After Step 3 increase to 70 C Prepare the total RNA 100yg in 100ul of mRNA Elution Buffer or 100ul of Nuclease Free water Note if the concentration of total RNA is less than 1pg pl The 100ug of RNA will have a volume large than 100ul In this case increase the volume of the Mag Bind mRNA Binding Buffer used in step 4 to equal the initial volume of the total RNA sample Swirl or shake the vial of Mag Bind Oligo dT magnetic beads until the particles are in a homogeneous suspension Heat the Total RNA sample to 65 C for 4 minutes Transfer 50ul of Mag Bind oligo dT magnetic beads into the total RNA sample Add 150ul of 2 x Mag Bind mRNA Binding Buffer mix by pipetting Incubate at 70 C for 3 minutes and then place at room temperature for 10 minutes Collect the magnetic beads by placing the tube on a magnetic separation de
6. se Solution S O RNA visible on RNase A not added to Add 1 vial of RNase to each bottle of agarose gel Solution I Solution I O ee o Plasmid DNA floats out of well Ethanol not completely Increase air dry time before elution while loading removed before elution step agarose gel Ooo foe fo Plasmid DNA will The DNA plate must be washed with Traces of ethanol remain not perform in absolute ethanol and dried before on column prior to elu i Paa downstream Hon elution Ethanol precipitation may be application required following elution 10
7. tocol Centrifugation Protocol Mag Bind mRNA Protocol Centrifugation Protocol Materials to be provided by user Microcentrifuge with adaptor for 1 5 ml tube Nuclease free 1 5ml centrifuge tubes Optional Absolute Ethanol chilled at 20 C Microcentrifuge Incubator capable of 70 C Before Starting Set an Incubator to 65 C After Step 3 increase to 70 C 1 Prepare the total RNA 100 9 in 100ul of mRNA Elution Buffer or 100ul of Nuclease Free water Note if the concentration of total RNA is less than 1 g l The 100 g RNA will have volume large than 100 I In this case increase the volume of the Mag Bind mRNA Binding Buffer used in step 4 to equal the initial volume of the Total RNA sample 2 Swirl or shake the vial of Mag Bind oligo dT magnetic beads until the particles are in a homogeneous suspension 3 Transfer 50ul of Mag Bind oligo dT magnetic beads into the Total RNA sample 4 Add 150ul of 2 x Mag Bind mRNA Binding Buffer mix by pipetting Incubate at 70 C for 3 minutes and then place at room temperature for 10 minutes 5 Centrifuge at 8 000 x g for 1 minute to collect the magnetic particles 6 Carefully remove the supernatant with a pipettor Avoid disturbing the magnetic particles pellet 7 Wash the magnetic beads by adding 200uI of mRNA Wash Buffer 8 Centrifuge at 8 000 x g for 1 minute to collect the magnetic particles 9 Carefully remove the supernatant with a pipettor Av
8. vice MSD 02 The liquid should be cleared after the magnetic beads are completely pelleted Aspirate the supernatant by pipetting Remove the tube from the magnetic stand Wash the magnetic beads again by adding 200ul of mRNA Wash Buffer Resuspend the magnetic beads by vortexing for 20 seconds Collect the magnetic beads by placing the tube on a magnetic separation device MSD 02 The liquid should be cleared after the magnetic beads are completely pelleted 10 11 12 13 14 Mag Bind mRNA Protocol Standard Protocol Aspirate the supernatant by pipetting Dry the magnetic beads pellet by air for 5 10 minutes Remove any liquid with a pipettor Remove the tube from the magnetic stand and then add 100ul of mRNA elution Buffer to the particles Incubate the tube at room temperature with gentle agitation for 5 minutes to release mRNA from the magnetic particles Place the tube on a magnetic stand to collect the magnetic particles Transfer the supernatant containing eluted mRNA into a RNase free tube The RNA can be store at 20 C for short term storage and 80 C for long term storage Optional Precipitate mRNA by ethanol precipitation Add 10ul of 5 M NaCl and 2 5 volume of cold absolute ethanol Incubate for 20 minutes at 20 C Centrifuge at maximum speed for 10 minutes at room temperature Wash once with 300ul of 70 ethanol and dissolve the purified mRNA with 10 20ul of nuclease free water Mag Bind mRNA Pro
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