Manual - Omega Bio-Tek
Contents
1. E Z N A system is stable for more than a year RNA Quality It is highly recommended that RNA quality be determined prior to all analyses The quality of RNA can be assessed by denaturing agarose gel electrophoresis and ethidium bromide staining Several sharp bands should appear on the gel These are the 28S and 18S ribosomal RNA bands as well as certain populations of MRNA and possibly viral RNA bands If these bands smear towards lower molecular weight RNAs then the RNA has undergone major degradation during preparation handling or storage RNA molecules less than 200 bases in length do not efficiently bind the HiBind matrix Page 8 of 10 Troubleshooting Guide Little or no RNA eluted Degraded RNA Problem in downstream applications DNA contamination RNA remains on the column Column is overloaded Bacterial cell wall is not completely removed Incomplete disruption or lysis of bacterial Source RNase contamination Salt carry over during elution Co purification of DNA Suggestion Repeat elution Pre heat DEPC water to 70 C prior to elution Incubate column at RT for 10 min with DEPC water prior to centrifugation Reduce amount of starting material e Use longer incubation time for lysozyme digestion or add more lysozyme e Use longer incubation time for lysozyme Increase centrifugation time Reduce amount of starting mater2. do DNase digestion for most downstream applications However certain sensitive RNA applications might require further DNA removal Follow the steps below for on membrane DNase I digestion See DNase I manual Product No E1091 for detailed information a For each HiBind RNA column prepare the DNase I digestion reaction mix as follows OBI DNase I Digestion Buffer RNase free DNase I 20 Kunitz unites ul Total volume Note 11 12 13 14 e DNase I is very sensitive and is subject to physical denaturation so do not vortex the DNase I mixture Mix gently by inverting the tube Prepare the fresh DNase I digestion mixture before RNA isolation e OBI DNase I digestion buffer is supplied with OBI RNase free DNase I set e Standard DNase buffers are not compatible with on membrane DNase I digestion b Dry column by spinning an additional 30 seconds then pipette 75 ul of the DNase I digestion reaction mix directly onto the surface of the HiBind RNA membrane in each column Make sure to pipette the DNase I digestion mixture directly onto the membrane DNase I digestion will not be complete if some of the mix sticks to the wall or the O ring of the HiBind RNA column c Incubate at room temperature 25 30 C for 15 minutes Place HiBind RNA Mini column in a clean 2 ml collection tube and add 500 ul RNA Wash Buffer I If on membrane DNase I digestion was performed in the previous step allow wash buf
3. Contents INGhOGUCEHION srs mac aes ating Bie ee ee eae Woe wa eee a 2 OVERVIEW mi kiam naa a Ante tM late SOAS aca Man ok is Ses te Rt he Tak 2 Storage and Stability 2 0 2 00 2 Kit Contents iy oe and aaa E Gates Pears eee Oe aa aes 3 Before Starting s meote dated ey asia dada egies alec te eae aes 3 E Z N A Bacterial RNA Spin Protocol 4 Vacuum Spin protocol 2 00 ee ee 6 Quantification and Storage of RNA 00000 ce aee 7 RNA Qualities Scent Saha ate Chae aie Woe Hated das 7 Troubleshooting Guide 1 ce a 8 Revised June 2008 Introduction E Z N A Bacterial RNA Kit allows rapid and reliable isolation of high quality total cellular RNA from a wide variety of bacterial species Up to 1 x 10 Bacterial cells can be processed The system combines the reversible nucleic acid binding properties of Omega Bio Tek s HiBind matrix with the speed and versatility of the spin column technology to yield approximately 50 100 ug of RNA There are no organic extractions thus reducing plastic waste and hands on time to allow multiple samples to be processed in parallel Purified RNA has Abs260 Abs280 ratios of 1 8 2 0 and is suitable for the following applications RT PCR e Northern Analysis e Differential display e Poly A RNA selection Overview If using the E Z N A Bacterial RNA Kit for the first time please read this booklet to become familiar with the procedures Bacter
4. fer to soak column at least 5 minutes before proceeding Centrifuge as above and discard flow through Place HiBind RNA Mini column in a new 2 ml collection tube provided Add 500 pl RNA Wash Buffer II and spin for 30 seconds at 10 000 x g Discard flow through and reuse the collection tube Add 500 pl RNA Wash Buffer II to column and centrifuge for 30 seconds at 8 000 10 000 x g to wash again Discard the flow through and reuse the collection tube Using the same collection tube dry the column by spinning for 2 minutes at 8000 10 000 xg Page 6 of 10 Note Drying the HiBind RNA Mini column is very important for removal of residual ethanol that will otherwise interfere with downstream applications 15 Transfer HiBind RNA Mini column to a new RNase free 1 5 ml collection tube not supplied and add 50 100 ul DEPC water directly onto the HiBind membrane Centrifuge for 1 minute at 8 000 10 000 x g to elute Repeat if the expected RNA yield is more than 60 ug Vacuum Spin Protocol V Spin Column Only Carry out lysis homogenization and loading onto HiBind RNA column as indicated in previous protocol Steps 1 8 Instead of continuing with centrifugation follow steps blow Note Please read through previous section of this manual before using this protocol 1 Prepare the vacuum manifold according to manufacturer s instructions and connect the HiBind RNA V Spin column to the manifold 2 Load the homogeni
5. ial e Follow protocol closely and work quickly Make sure that 2 mercaptoethanol is added to BRK Lysis Buffer e Ensure not to introduce RNase during the procedure e Check buffers for RNase contamination e Ensure Wash Buffer II has been diluted with 96 100 ethanol as indicated on bottle e Diluted Wash Buffer II must be stored at room temperature e Repeat wash with Wash Buffer II e Digest with RNase free DNase I and inactivate at 75 C for 5 min Low Abs ratios Page 10 of 10 RNA diluted in acidic buffer or water DEPC treated water is acidic and can dramatically lower Abs260 values Use TE buffer pH 8 to dilute RNA prior to analysis
6. ial cells are grown to log phase and harvested Bacterial cell walls are removed by lysozyme digestion Following lysis binding conditions are adjusted and the samples are applied to HiBind RNA spin columns Two rapid wash steps remove trace salt and protein contaminants and RNA is eluted in water or low ionic strength buffer Purified RNA can be directly used in downstream applications without the need for further purification Storage and Stability All components of the E Z N A Bacterial RNA Kit are stable for at least 24 months from the date of purchase when stored at 22 C 25 C During shipment or storage in cool ambient conditions precipitates may form in Buffer BRK It is possible to dissolve such deposits by warming the solution at 37 C New This Edition A heat incubation step has been added to reduce the amount of co purification of DNA Page 2 of 10 Kit Contents RNA Wash Buffer II 5 ml Glass Beads 200 mg Lysozyme 8 mg 80 mg 4 x 80 mg DEPC Water 1 5 ml User Manual 1 Product Number HiBind RNA Mini column 2 ml Collection Tubes RNA Wash Buffer I Buffer BRK contains a chaotropic salt Use gloves and protective eyeware when handling with this solution Before Starting Please take a few minutes to read this booklet thoroughly to become familiar with the protocol Prepare all materials required before starting procedure to minimize RNA degradation Prepare a stock solution of lysozy
7. me provided at 15 mg ml with TE buffer and aliquot into adequate portions Store aliquots at 20 C Bacterial should be harvested in log phase growth B mercaptoethanol B ME must be added to Buffer BRK before use This mixture can be stored for 1 month at room temperature Dilute RNA Wash Buffer II Concentrate with ethanol as follows and store at room temperature R6950 00 Add 20 ml absolute ethanol 96 100 l R6950 01 Add 48 ml absolute ethanol 96 100 R6950 02 Add 200 ml absolute ethanol 96 100 per bottle R6950 00 R6950 01 R6950 02 i Purification 5preps 5preps 50 Preps 200 Preps SEES BRK Lysis Buffer 20 ml 80 ml l Bacterial RNA Spin Protocol Materials supplied by users e Tabletop microcentrifuge and RNase free 2 0 or 1 5 ml tubes e Absolute ethanol 96 100 do not use other alcohols e Waterbath or Incubator set to 70 C This method allows bacterial RNA isolation from up to 3 ml LB culture 1 Grow Bacteria in LB media to log phase Do not use overnight culture 2 Harvest no more than 3 ml culture lt 5 x 10 bacteria by centrifugation at 4 000 5000 x g for 5 10 min at 4 C 3 Discard medium and resuspend cells in 100ul Lysozyme TE Buffer Mix by vortexing at maxi speed for 30 seconds Note The amount of enzyme required and or the incubation time may need to be modified depending on the bacterial strain used Complete digestion of the cell wall is e
8. ssential for efficient lysis For some bacteria other enzymes may be more effective 4 Incubate at 30 C for 10 minutes Incubate on a shaker incubator or vortex for 20 seconds for every 2 minutes during incubation 5 Add 350 ul BRK lysis buffer and 25 40 mg glass beads to the sample and vortex vigorously for 5 minutes Centrifuge for 5 minutes at maximum speed in a micro centrifuge Note Ensure B mercaptoethanol B ME is added to BRK Lysis Buffer 20 pIl ml before use 6 Transfer 400pI of the supernatant into a new 1 5 ml centrifuge tube Page 4 of 10 10 11 Incubate sample at 70 C for 5 minutes Centrifuge at maximum speed gt 13 000 x g for 2 minutes Transfer the supernatant into a new 1 5 ml tube not supplied Add 280 ul absolute ethanol 96 100 to the lysate and mix well by vortexing at maxi speed for 15 seconds Apply sample including any precipitate that may have formed to a HiBind RNA mini column inserted in a 2 ml collection tube Centrifuge for 30 seconds at 8 000 10 000 x g Reuse the collection tube for next step Add 400ul RNA Wash Buffer I to the column Centrifuge at 10 000 x g for 2 minutes Discard the flow through and collection tube If on membrane DNase I digestion is desired proceed to Step 11 otherwise go to Step 13 DNase I Digestion Optional Since HiBind RNA resin and spin column technology actually removes most of DNA without the DNase I treatment it is not necessary to
9. zed sample into HiBind RNA V spin column 3 Switch on vacuum source to draw the sample through the column 4 Optional Perform on membrane DNase I digestion steps if sensitive downstream application is desired See Step 10 Pages 5 6 above 5 Wash the column by adding 500 ul RNA Wash Buffer I Draw the wash buffer through the column by turning on the vacuum source 6 Wash the column by adding 500 ul RNA Wash Buffer II Draw the wash buffer through the column by turning on the vacuum source 7 Insert the column into a 2 ml collection tube and transfer the column to a micro centrifuge Spin for 1 minute to dry the column 8 Place the column in a clean 1 5 ml micro centrifuge tube and add 50 100ul DEPC water Stand for 1 2 minute and centrifuge for 1 minute to elute RNA Quantification and Storage of RNA To determine the concentration and purity of RNA measure absorbency at 260 nm and 280 nm in a spectrophotometer 1 O D unit measured at 260 nm corresponds to 40 ug of RNA per ml The ratio of A Azgo Of pure nucleic acids is 2 0 while for pure protein it is approximately 0 6 A ratio of 1 8 2 0 corresponds to 90 100 pure nucleic acid Phenol has an absorbency maximum at 275 nm and can interfere with spectrophotometric analysis of DNA or RNA However the E Z N A Bacterial RNA Kit eliminates the use of phenol and avoids this problem Store RNA samples at 70 C in water Under such conditions RNA prepared with the
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