Contents Introduction Overview Storage and Stability Binding Capacity
Contents
1. Contents INtrOdUCTION ce sa crer eae eee Bee Seale Pee eee REO AD eee oa eee 2 2 OVEIVIEW si 0 6 Shenae ele ba ate edn Sade ed ed ee tg eh a oe Bag 2 Storage and Stability 2 2 0 0 0 0 a a a ee 2 Binding Capacitysz rs nenene BE a tn tie Wachee eer Aad a ttl 2 Kit Contents sic ssn ae i a Pe ee eee Eee ieee ee 3 Before Starting as as anes tee ce oe a had acd a a ted BE OA ee Pe 4 E Z N A 2 Yeast RNA Kit Spin Protocol 0 0 0 0000s cece eee 5 Quantitation and Storage of RNA 2 0 0 0 eee 7 RNA Quality eeun a agar thie hia Mates el eee 7 Troubleshooting Guide 0 00 cee eee 8 Page 1 of 8 Introduction The E Z N A Yeast RNA Kit allows convenient isolation of high quality total RNA from a wide variety of yeast species Up to 2 x 10 log phase cultured yeast cells can be processed The system combines the reversible nucleic acid binding properties of HiBind matrix with the speed and versatility of spin column technology to yield approximately 30 ug of RNA with an Azso Azs0 ratio of 1 7 1 9 Purified RNA is suitable for downstream applications such as RT PCR DD PCR and hybridization techniques There are no organic extractions thus reducing plastic waste and hands on time to allow multiple samples to be processed in parallel Overview If working with RNA for the first time please read this booklet to become familiar with the procedure Yeast cells are grown to log phase and spheroblasts are sub2. an equal volume 70 ethanol to the lysate and mix thoroughly by pipetting A white precipitate may form upon addition of ethanol it will not interfere with the procedure Do not centrifuge the tube Apply the entire sample around 700 ul to a HiBind RNA Mini column assembled in a 2 ml collection tube supplied Centrifuge at gt 10 000 x g for 30 60 seconds at room temperature Discard flow through and re use collection tube in Step 8 or Step 9 Note This is the point at which optional on membrane DNase digestion should begin If DNase digestion is desired skip Step 8 and proceed to Step 9 If DNase digestion is not needed proceed with Step 8 Wash the sample by adding 700ul of RNA Wash Buffer to the column Centrifuge at 10 000 x g for 30 60 seconds at room temperature Discard the flow through and re use the collection tube DNase Digestion Optional Page 5of 8 10 Since HiBind RNA resin and spin column technology actually removes most DNA without the DNase treatment this DNase digestion step is not necessary for most downstream applications However certain sensitive RNA applications might require further DNA removal See DNase Cat E1091 for detailed information a Wash the column by adding 300uI of RNA Wash Buffer to the column Centrifuge at 10 000 x g for 30 60 seconds at room temperature Discard the flow through and re use the collection tube b For each HiBind RNA column have the follow
3. 000 x g for 30 60 seconds and discard flow through Re use the collection tube in next step Note RNA Wash Buffer II Concentrate must be diluted with absolute ethanol Page 6 of 8 before use Refer to Page 4 or to label on bottle for directions 11 Wash sample with a second 500 ul of RNA Wash Buffer II and centrifuge and discard flow through as in preceding step Using the same collection tube centrifuge the spin cartridge at 10 000 x g for 2 min at full speed to completely dry the HiBind matrix 12 Elution of RNA Transfer the column to a clean 1 5 ml microfuge tube not supplied with kit and elute the RNA with 30 50 ul of DEPC treated water supplied with kit Make sure to add water directly onto column matrix Centrifuge 1 min at maximum speed A second elution may be necessary if the expected yield of RNA is greater than 50 ug Alternatively RNA may be eluted with a greater volume of water While additional elutions increase total RNA yield the concentration will be lowered since more than 80 of RNA is recovered with the first elution Pre heating the water to 70 C before adding to column and incubating column 5 min at room temperature before centrifugation may increase yields Quantitation and Storage of RNA To determine the concentration and purity of RNA measure absorbance 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 DEPC water is slightly acidic and can d
4. 96 100 ethanol R6870 01 Add 48 ml absolute 96 100 ethanol R6870 02 Add 48 ml absolute 96 100 ethanol to each bottle E Z N A Yeast RNA Kit Spin Protocol Page 4of 8 This protocol uses enzymatic lysis to prepare spheroblasts Have the following reagents and supplies ready While the HiBind RNA mini columns can bind up to 100 ug RNA for effective purification use no more than 2 x 10 log phase yeast cells For S cerevisiae grown in YPD an OD of 1 0 corresponds to approximately 2 x 10 cells per ml 1 Collect no more than 2 x 10 yeast cells in a 15 ml tube by centrifuging for 5 min at 1000 x g at 4 c NOTE Use only freshly harvested cells for preparation of spheroblasts Aspirate and discard supernatant completely and resuspend cells in 2 0 ml Buffer SE Lyticase mixture Incubate at 30 C for 30 min inverting the tube once every 10 min Pellet spheroblasts by centrifuging 5 min at 400 x g at room temperature Carefully aspirate and discard supernatant Incomplete removal of supernatant will prevent complete lysis of spheroblasts in the next step Add 350 ul of Buffer YRL 2 mercaptoethanol and 50 mg glass beads into the tube Vortex at maxi speed for 5 minutes to lyse and homogenize the sample If bead mill is available lyse the cell with bead mill at top speed until the cells are completely lysed Centrifuge at 13 000 x g for 3 min Transfer the supernatant into a new centrifuge tube Add
5. ing DNase digestion reaction mixture prepared in advance as follows OBI DNase Digestion Buffer 73 5 pl RNase free DNase 20 Kunitz unites ul 1 5 ul Tota volume Total volume 75 ul Note 1 DNase lis very sensitive and is thus subject to physical denaturation so do not vortex this DNase mixture Mix gently by inverting the tube Prepare the fresh DNase digestion mixture before RNA isolation 2 OBI DNase digestion buffer is supplied with the OBI RNase free DNase set 3 Standard DNase buffers may not be compatible with the Omega Bio Tek Kit for on membrane DNase digestion c Dry column by spinning an additional 30 seconds then pipet 75 ul of the DNase digestion reaction mixture directly onto the surface of the HiBind RNA membrane in each column Make sure to pipet the DNase digestion mixture directly onto the membrane DNase digestion might not be complete if some of the mixture sticks to the wall or to the O ring of the HiBind RNA column d Incubate at room temperature 25 30 C for 15 minutes d Place column in a clean 2 ml collection tube and add 400 ul RNA Wash Buffer Allow wash buffer to soak column for at least 5 minutes before proceeding Centrifuge at 10 000 x g for 15 seconds at room temperature Discard flow through and re use the collection tube in next step Place column in the same 2 ml collection tube and add 500 uI RNA Wash Buffer II diluted with ethanol Centrifuge at 10
6. ramatically lower absorbance values We suggest that you dilute the sample in a buffered solution TE for spectrophotometric analysis The ratio of A o A 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 Store RNA samples at 70 C in water Under such conditions RNA prepared with the 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 best be assessed by denaturing agarose gel electrophoresis and ethidium bromide staining Two sharp bands should appear on the gel These are the 28S and 18S ribosomal RNA bands If these band smear towards lower molecular weight RNAs then the RNA has undergone major degradation during preparation handling or storage Troubleshooting Guide Page 7 of 8 Little or no RNA eluted Clogged column Degraded RNA Problem in downstream applications DNA contamination Low Abs ratios Page 8 of 8 RNA remains on the column Incomplete spheroblasting Source RNase contamination Salt carry over during elution RNA diluted in acidic buffer or DEPC water Repeat elution Pre heat DEPC water to 70 C prior to elution Incubate column for 5 min with water prior to centrifugation Extend incubation with Lyticase Increase Lyticase to 100 units per 10 cells Reduce amount of s
7. sequently prepared using Buffer SE and lyticase Following lysis binding conditions are adjusted and the sample is applied to a HiBind RNA spin column Two rapid wash steps remove trace salt and protein contaminants and RNA is eluted in DEPC treated water Purified RNA can be directly used in downstream applications without need for further purification Storage and Stability All components of the E Z N A Yeast 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 YRL These precipitates should be dissolved by warming the solution at 37 C and gently shaking its container Binding Capacity Each HiBind RNA Mini Column can bind up to 100 ug Total RNA Use no more than 3 ml log phase yeast culture is not recommended Page 2 of 8 Kit Contents Product Number R6870 00 R6870 01 R6870 02 Purification times 5 Preps 50 Preps 200 Preps HiBind RNA Mini column 5 50 200 2 ml Collection Tubes 15 150 600 Glass Beads 250mg 2 5g 10g Buffer SE 15 ml 120 ml 2x 210 ml Buffer YRL 5 ml 20 ml 80 ml RNA Wash Buffer 5 ml 50 ml 3x 70m RNA Wash Buffer II 2ml 12 ml 4x12 ml Lyticase 500 units 5000 units 4 x 5000 DEPC water 1ml 10 ml 40 ml User Manual 1 1 1 Buffer YRL contains a chaotropic salt Use CAUTION gloves and protective eye wear when handling this solution 2 Materials
8. tarting material Use only freshly harvested cells Do not store cells prior to extraction unless they are lysed with Buffer YRL first Follow protocol closely and worl quickly Ensure not to introduce RNase during the procedure Check buffers for RNase contamination Ensure Wash Buffer II Concentrate has been diluted with 4 volumes of 100 ethanol as indicated on bottle Wash Buffer II must be stored and used at room temperature Repeat wash with Wash Buffer II Performthe optional DNase digestion step or Digest with RNase free DNase after elution and inactivate at 75 C for 5 min DEPC treated water is acidic and can dramatically lower Abs260 values Use TE buffer at pH 8 0 to dilute RNA prior to spectrophotometric analysis
9. to Be Provided by User Microcentrifuge and nuclease free 2 0 and 1 5 ml tubes e Swinging bucket centrifuge and sterile 15 ml tubes e Waterbath equilibrated to 30 C e 70 ethanol do not use other alcohols e Absolute ethanol 96 100 e 2 Mercaptoethanol Before Starting Page 3 of 8 e Whenever working with RNA always wear latex gloves to minimize RNase contamination Use only clean RNase free disposable plastic pipette tips when using the supplied reagents During the procedure work carefully but quickly e Under cool ambient conditions crystals may form in Buffer YRL This is normal and the bottle should be warmed to redissolve the salt e 2 mercaptoethanol f mercaptoethanol is key in denaturing RNases and must be added to an aliquot of Buffer YRL before use Add 20 ul of 2 mercaptoethanol per 1 ml of Buffer YRL This mixture can be stored for 1 month at room temperature Prepare Buffer SE Lyticase Mixture R6870 00 Dissolve vial of lyticase in 10 ml SE Buffer and add 10ul of 2 mercaptoethanol per 1 ml of Buffer SE R6870 01 Dissolve vial of lyticase in 100 ml SE Buffer and add 10ul of 2 mercaptoethanol per 1 ml of Buffer SE R6870 02 Dissolve vial of lyticase in 400 ml SE Buffer and add 10ul of 2 mercaptoethanol per 1 ml of Buffer SE RNA Wash Buffer II is supplied as a concentrate and must be diluted with absolute ethanol as follows and stored at room temperature R6870 00 Add 8 ml absolute
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