RNaid® Kit - MP Biomedicals
Contents
1. c cesses Kit Components and User Supplied Materials 2 1 RNaid Kit Component cscs 2 2 User Supplied Materials c ccccccscssessseeee Important Considerations Before Use 0008 ROA GG anA Safety Precautions PROG O eea tan temeuntumtasetanteetnte 6 1 Isolation of RNA from Solutions from Agarose or Polyacrylamide Gels Containing up to 6M Urea 6 2 Isolation of RNA from Agarose Gels Containing Formaldehyde aenn 6 3 Purification of RNA from Transcription Reactions Troubleshooting Guide eerren 7 1 Gel icicle mre tT ert 7 2 Adsorption to RNAMATRIX c ccccesseseeseeees 7 3 Spectrophotometer Readings Recommended Reference Format for Publication Related Products ccccsccccccccceeeeeeeeeeeeeeaeaeeeeess Product Use Limitation amp Warranty www mpbio com O 9 T 9 1 Introduction to the RNaid Kit The RNaid Kit contains all of the solutions and reagents except for ethanol necessary for the isolation and purification of RNA in twenty minutes from agarose and polyacrylamide gels from solutions and from unincorporated radioactive label see Protocol The resulting RNA is suitable as a substrate for multiple enzymatic manipulations including reverse transcription RNase protection assays and in vitro translation For isolation of total RNA from tissues cells use the RNaid Plus Kit Cat 1109 200 or Total RNA SAFEKIT Cat 1008 200 For2. However separation properties of polyacrylamide are usually superior To improve recovery efficiency from polyacrylamide extract RNA at elevated temperature 55 65 C and extended time 20 min Both formaldehyde and agarose gels as well as TBE polyacrylamide gels create unfavorable binding conditions for RNA to RNAMATRIX as the pH increases upon dissociation of the gel Adjustment to more acidic conditions is necessary In both cases optimal binding efficiency can be restored by adjusting the pH to an appropriate range using 10 acetic acid kit supplied When using formaldehyde do not exceed a final concentration of 6 6 in the gel and buffer and prepare all solutions fresh just before use Avoid adding ethidium bromide to the formaldehyde gel since it will fluoresce and render the detection of RNA bands extremely difficult Instead add ethidium bromide to the RNA sample before loading as described in Section 7 3 below Always remember to process RNA as quickly as possible never leave RNA inside gel for long periods of time gt 1hr remove immediately and store under proper conditions to avoid degradation Important Note Agarose or acrylamide used for separation of RNA should be of highest purity available The quality of chemicals used for gel buffers is equally critical for good separation results When preparing urea solutions always filter purify through 0 45 um membrane to remove insoluble particles Pre treat hardware such
3. 10 mM each 4 ul radioactively labeled rCTP 200 pCi 8 ul distilled water 1 ul DNA template 1ug ul 1 ul RNA Polymerase i e T3 T7 or Sp6 Polymerase 25 ul total reaction volume 2 Mix and incubate at 37 C for 1 hour When completed remove 1 ul of the reaction and determine TCA precipitable counts to calculate incorporation Removal of DNA Template and Hydrolysis of RNA Note Depending on the size of the transcribed RNA and the purpose of use it may be necessary to shorten the transcripts by hydrolysis with sodium hydroxide After hydrolysis it is crucial to neutralize the pH before purification of the RNA with RNAMATRIX If the pH of the sample is alkaline the RNA will not adsorb to the matrix 3 Add 1 ul of RNase free DNase 10 units ul Incubate at 37 C for 10 minutes and place on ice If probe is to be hydrolyzed continue with Step 4 Otherwise skip ahead to Purification of RNA Step 7 below 4 Add 50 ul ETS buffer 10 mM Tris HCl pH 7 5 1 mM EDTA 0 1 SDS 1 7 ul 5M NaCl and 1 ul 1M DTT Mix thoroughly 5 Add 10 ul 2N NaOH for hydrolysis of RNA transcripts Incubate on ice for 30 minutes for smaller transcripts lt 1kb or for 60 minutes for larger transcripts gt 1 kb As an example a 1 kb transcript can be shortened to 150 220 bases by incubating 30 40 minutes at 4 C 6 Warm to room temperature then add 20 ul 2x volume of 1 M MES buffer to neutralize pH and stop hydrolysis Add MES buffer after warm
4. the sample Small aggregates will not affect the procedure Problem During adsorption to RNAMATRIX not all RNA is bound and removed from solution Solution The amount of RNAMATRIX was not sufficient to adsorb all of the RNA Transfer supernatant with unbound RNA to new vial add 5 10 ul of new RNAMATRIX You may also re use the matrix previously applied to this sample Save supernatant until after the initially bound RNA is eluted from the matrix then add supernatant back to the same matrix Incubate and follow wash procedures as described Elute RNA with 5 10 ul DEPC Treated Water and combine with RNA eluted from the first adsorption process 7 3 Spectrophotometer Readings Technical Background The amount of nucleic acid in the eluent from RNAMATRIX can be determined by optical density at 260 nm The RNAMATRIX material absorbs over a wide range of the spectrum including UV This absorption is consistent and very low over a range from 210 nm to approximately 310 nm absorbing 0 01 OD units Residual matrix material can easily be removed by centrifugation and this will not contribute to any absorption readings Problem 0D260 measurement of the RNA Wash Solution supernatant shows UV absorption Solution Spin vial with pellet of RNA RNAMATRIX complex for 10 seconds and remove traces of liquid with a small bore pipet tip Resuspend pellet in 500 ul RNA Wash Solution and mix with pipet tip to resuspend the matrix evenly Spin for
5. 1 minute in microcentrifuge at maximum speed Remove supernatant and measure OD260 If absorption is still above background repeat washing procedure If no absorption is detectable proceed with elution of the RNA as described Problem Eluted RNA is contaminated with residual RNAMATRIX Solution Small amounts of RNAMATRIX in the RNA sample are inert and will not effect enzymatic reactions However RNAMATRIX absorbs UV light and might effect 0D260 280 measurements To remove residual RNAMATRIX from the eluted RNA re spin vial in microcentrifuge for 30 seconds at maximum speed and remove RNA solution with small bore pipet tip Be careful not to touch bottom of tube near pellet www mpbio com 15 RNaid Kit area For consistent and reliable measurement of multiple samples complete removal of residual RNAMATRIX particles can be accomplished by centrifugal filtration through a SPIN Filter unit or by passing RNA sample through a sterile 0 2 um membrane attached to a syringe 8 Recommended Reference Format for Publications RNA was isolated from specific sample using the RNaid Kit MP Biomedicals Santa Ana CA 9 Related Products Description Size Catalog FastPrep 24 Instrument 6004 500 FastPrep Instrument 001 100 FastPrep FP120A Instrument 001 120 FastPrep FP220A Instrument 001 220 FastDNA Kit 540 400 FastDNA SPIN Kit 540 600 FastDNA SPIN Kit for Soil 560 200 FastDNA 50ml SPIN Kit for Soil
6. 570 200 FastRNA Pro Soil Direct Kit 070 050 FastRNA Pro Soil Indirect Kit 075 050 FastRNA Pro Red Kit Yeast amp Fungus 035 050 FastRNA Pro Green Kit Plant amp Animal 045 050 FastRNA Pro Blue Kit Bacteria 025 050 FastProtein Blue Matrix 6550 400 Fd O Io O gt www mpbio com 10 Product Use Limitation amp Warranty The products presented in this instruction manual are for research or manufacturing use only They are not to be used as drugs or medical devices in order to diagnose cure mitigate treat or prevent diseases in humans or animals either as part of an accepted course of therapy or in experimental clinical investigation These products are not to be used as food food additives or general household items Purchase of MP Biomedicals products does not grant rights to reproduce modify or repackage the products or any derivative thereof to third parties MP Biomedicals makes no warranty of any kind expressed or implied including merchantability or fitness for any particular purpose except that the products sold will meet our specifications at the time of delivery Buyer s exclusive remedy and the sole liability of MP Biomedicals hereunder shall be limited to at Our discretion no replacement or compensation product credits refund of the purchase price of or the replacement of materials that do not meet our specification By acceptance of the product Buyer indemnifies and holds MP Biomedic
7. Acetic Acid per every 0 5 ml of liquid to change pH to 5 0 5 5 check with pH paper This will increase recovery efficiency Estimate the amount of RNA expected and add 1 ul of RNAMATRIX for every ug of RNA Add a minimum of 5 ul of RNAMATRIX Mix well and allow binding of RNA to the matrix for at least 5 minutes at room temperature Mix occasionally to keep RNAMATRIX in suspension during adsorption Spin for 1 minute in microcentrifuge at maximum speed to pellet RNA RNAMATRIX complex Remove supernatant and save aside if supernatant contains residual RNA more RNAMATRIX can be added for complete recovery Spin pellet again briefly and remove residual liquid with small bore pipet tip Add 500 ul RNA Wash solution remember to add ethanol before first use see Section 3 above and resuspend pellet by mixing with pipet tip Spin for 1 minute in microcentrifuge at maximum speed and remove supernatant Repeat washing Step 5 at least 1 2 times After last wash spin tube again briefly and remove residual liquid with small bore pipet tip Resuspend pellet in RNase free water use kit supplied DEPC Treated Water Use 10 20 ul per 5 ul RNAMATRIX Mix thoroughly with pipet tip and elute RNA by incubating at 45 55 C for 5 minutes Spin for 2 minutes in microcentrifuge at maximum speed The RNA will be in the supernatant Remove supernatant containing RNA to sterile vial Note Before spectrophotometric analysis of the sample spin tube for
8. difficult to lyse samples use the FastRNA Kits visit www mpbio com for information The RNaid Kit provides sufficient reagents for two hundred or more RNA purifications 2 Kit Components and User Supplied Materials 2 1 RNaid Kit Components DEPC Treated Water 15 ml RNA Binding Salt 60 ml RNA Wash Concentrate 120 ml RNAMATRIX 1 5 ml 10 Acetic Acid 0 5 ml User manual 1 each MSDS Online www mpbio com 1 each Certificate of Analysis 1 each 2 2 User Supplied Materials Microcentrifuge that can freely spin 2 0 ml tubes Microcentrifuge tubes 2 0 ml and 1 5 ml Rotator or low speed vortex Gel Electrophoresis Apparatus Power Source Sterile water TAE Buffer 100 Ethanol www mpbio com 5 RNaid Kit Note The User Supplied Materials below are necessary dependent upon your experimental approach See protocols below for more information Citric acid 8M Urea Formamide MOPS EDTA NaOAc Formaldehyde Ethidium bromide Transcription Reaction Kit 3 Important Considerations Before Use The RNaid Kit and all of its reagents are for research use only All kit components have been lot qualified for the isolation of undegraded RNA To avoid contamination of samples and reagents with RNase follow appropriate laboratory procedures wear surgical or similar gloves use sterile vials pipets and pipet tips etc To prepare glass ceramic or metal homogenizers clean and bake at 200 C for 2 hours to remove any
9. 1 minute to pellet any remaining RNAMATRIX material and remove RNA sample to new vial Alternatively pass RNA sample through a sterile 0 2 um membrane syringe filter attached to a syringe or in a spin column g 8 Optional Repeat elution Steps 7 and 8 to recover an additional 5 15 RNA www mpbio com 6 2 Isolation of RNA from Agarose Gels Containing Formaldehyde Note Reagents for agarose formaldehyde gels are not included with the kit 10x Gel Buffer 200 mM MOPS pH 7 0 adjust with NaOH 10 mM EDTA 10 mM NaOAc Prepare 1 2 agarose gel containing 6 6 formaldehyde and 1X gel buffer Do not add ethidium bromide to the gel only to the RNA sample as described below Run gel at 3 5 V cm in 1X gel buffer with 6 6 formaldehyde at pH 7 0 Preparation of RNA sample for gel 10 ul formamide deionized 4 ul formaldehyde 37 12 3 M 2 ul 10x gel buffer 3 ul RNA up to 20 ug 1 ul ethidium bromide 400 ug ml Heat at 65 C for 10 minutes before loading in well of agarose gel Formamide is dense enough to allow the sample to be loaded without adding an additional dense liquid however a loading dye mixture can be used if preferred Isolation of RNA from Agarose Formaldehyde Gel 1 Excise RNA band s from gel after electrophoresis Visualize RNA with long wave UV for minimal length of time while cutting gel Determine approximate volume of gel slice s by weight and place slice s into microcentrifuge tubes 2 Adju
10. 2 times Re spin and remove traces of liquid as described in Step 5 Resuspend pellet completely in DEPC Treated Water by mixing with pipet tip Use 10 20 ul of water per 5 ul RNAMATRIX Elute RNA by incubating at 80 C for 10 minutes Spin tube for 2 minutes and remove supernatant with RNA If using the SPIN Filter option transfer suspension to a SPIN Filter and spin for 1 minute in microcentrifuge The supernatant containing RNA will be in the catch tube Optional A second elution will yield 5 15 more RNA Heat eluted RNA to 80 C for 10 minutes to further dissociate residual formaldehyde from RNA This second heating step will reverse chemical modification of the RNA caused by formaldehyde Boedtker H 1967 Biochemistry 6 2718 2727 and will render RNA biologically active as substrate for modifying enzymes Let cool to room temperature to allow RNA to renature or place on ice immediately to avoid renaturing The RNA is now ready for use in enzymatic manipulations 6 3 Purification of RNA from Transcription Reactions Note Reagents for transcription template removal and hydrolysis are not included with the kit Transcription Reaction Commercially available as a kit from several manufacturers i Combine the following 5 ul 5x transcription buffer 200 mM Tris HCI pH 7 5 at 37 C 30 mM MgCl2 www mpbio com 50 mM NaCl 10 mM spermidine 1 ul RNase Inhibitor 1 unit ul 1 ul 50 mM DTT 1 ul each rATP rGTP rUTP
11. IX Technical Background The conditions for binding of RNA to the RNAMATRIX can be varied according to lysis and or purification method see Gel Purification section and desired RNA population or size of RNA molecules The binding efficiency is influenced by ionic strength and pH Adsorption in the presence of guanidine thiocyanate only lysis solution selects for larger RNA s Addition of RNA Binding Salt during adsorption causes binding of smaller molecules Optimal pH range for adsorption of RNA to RNAMATRIX is Ph 5 5 7 5 higher pH conditions result in a loss of binding capacity In general lower pH 5 5 6 0 tends to increase binding of smaller RNA molecules slightly higher pH 6 5 7 5 favors larger sizes Problem After addition of RNAMATRIX and mixing the matrix forms aggregates and does not disperse evenly throughout the sample Solution Aggregation of the RNAMATRIX tends to occur at high concentrations of nucleic acid Try breaking up aggregates with pipet tip or by rocking tube back and forth quickly Small aggregates will not effect the procedure Problem During RNA wash steps of the pellet the RNAMATRIX does not disperse evenly Solution The pellet is as easily dispersed in the RNA Wash solution containing ethanol as in water When adding the RNA Wash solution to the pellet use same pipet tip and 14 www mpbio com break up pellet by stirring Be careful to expel any trapped matrix from the tip back into
12. Instruction Manual RNaid Kit Isolates and Purifies RNA from Agarose or Polyacrylamide Gels from Solutions and from Unincorporated Radioactive Label One Call ame 200 P One Source ii gt A World of p Storage Ambient 15 30 C Biotechnology Catalog 1007 200 Reagents Revision 1007 200 100CT n A Sae www mpbio com MP Biomedicals 29525 Fountain Parkway Solon OH 44139 tel 1 800 854 0530 fax 1 800 334 6999 RNaid Kit Lyse BIG and FAST with Interchangeable Adapters CoolTeenPrep Adapter 6x15ml samples under QuickPrep Adapter 24 x 2 ml samples included with cryogenic conditions FastPrep 24 Instrument CoolBigPrep TallPrep Adapter Adapter 2 x 50ml samples under i 24 x 4 5ml samples cryogenic conditions TO a CoolPrep TeenPrep Adapter Adapter 24 x 2 ml samples a 12x 15ml samples under temperature controlled conditions HiPrep Adapter 48 x 2ml samples BigPrep Adapter 2 x 50 ml samples a The FastPrep 24 instrument is delivered with the QuickPrep Adapter www mpbio com RNaid Kit Isolates and Purifies RNA from Agarose or Polyacrylamide Gels from Solutions and from Unincorporated Radioactive Label 200 Preps storage Ambient 15 30 C Catalog 1007 200 Revision 1007 200 100CT www mpbio com RNaid Kit TABLE OF CONTENTS SS a Oe Introduction to the RNaid Kit
13. als harmless against and assumes all liability for the consequence of its use or misuse by the Buyer its employees or others including but not limited to the cost of handling Said refund or replacement is conditioned on Buyer notifying within thirty 30 days of receipt of product Failure of Buyer to give said notice within thirty 30 days shall constitute a waiver by the Buyer of all claims hereunder with respect to said material s FastDNA FastRNA FastPrep QBiogene and BIO 101 Systems are registered trademarks of MP Biomedicals LLC www mpbio com 17 RNaid Kit Take Advantage of FastPrep Kits Ready to use Protocols For DNA RNA And Protein Isolation From Any Sample e Rapid and reproducible sample lysis and purification process e No cross contamination with the closed lysing matrix tubes e Increased yields of high quality DNA RNA and proteins e Integrity and size of DNA RNA and proteins are retained e Nucleic acids and proteins are ready to use in downstream application DNA f PROTEIN FastPrep Kits RNA FastDNA Kit and FastDNA Spin Kit Cat N 6540 400 Cat N 6540 600 respectively 100 preps e Lyse and isolate DNA in less than 30 minutes e Plant animal yeast fungal and microbial samples e No hazardous organic reagents required e SPIN filters streamline silica handling FastDNA Spin Kit FastDNA Spin Kit for Soil Cat N 6560 200 100 preps e Lyse and isolate DNA in less
14. as glassware gel devices combs spacers etc with RNase Erase to remove contaminating RNases Use Sterile individually wrapped and unopened disposable supplies if possible Prepare all buffers and gels with DEPC Treated Water and autoclaved buffers Problem Agarose gel slice does not dissolve completely Solution Follow instructions and incubate agarose at 45 55 C for 10 minutes If gel has not dissociated add more RNA Binding Salt and continue incubation at same temperature for 10 minutes www mpbio com 13 RNaid Kit Problem Liquid cannot be completely removed from polyacrylamide gel elution or transferred liquid contain small pieces of polyacrylamide Solution Remove gel pieces by forcing liquid trough a 0 2 um membrane attached to a syringe Acrodisc PF with 0 2 um Supor and 0 8 um Pre filter works well supplied sterile by Gelman Sciences Alternatively use a large pipet tip with a sterile cotton plug Do not use glass wool Problem The RNA solution eluted from the gel has been checked with pH paper and shows a pH gt 7 5 Solution Use the kit supplied 10 Acetic Acid to adjust pH Add 2 ul 10 Acetic Acid per every 0 5 ml of eluted liquid to change pH to 5 0 6 0 and check with pH paper This procedure should be used for both TBE polyacrylamide and formaldehyde agarose eluents Once pH has been adjusted to optimal range continue with RNA purification and add RNAMATRIX to the eluted RNA 7 2 Adsorption to RNAMATR
15. beled RNA to new tube If using the SPIN option transfer suspension to a SPIN Filter and spin for 1 minute in microcentrifuge The supernatant containing RNA will be in the catch tube Optional A second elution will yield 5 15 more RNA Note If the RNAMATRIX retains radioactive label elute a second time by resuspending pellet in RNase free water and incubating at 80 C for 3 minutes Spin and remove supernatant to new tube If the second elution does not contain any radioactive label and the RNAMATRIX still retains radioactivity all RNA has been eluted Radioactivity retained by the RNAMATRIX represents reagent decay products which bind to the RNAMATRIX If available use fresh radioactive reagents no older than two weeks from the date of manufacture since the amount of decay products increases with age of radioactive materials especially 32P labelled reagents It is important to purify radioactively labeled RNA using the RNaid kit with SPIN immediately after transcription reaction is complete to minimize binding of RNA radiolysis products to the RNAMATRIX www mpbio com 7 Trouble Shooting Guide 7 1 Gel Purification Technical Background The right choice of gel separation system is extremely important for best results Criteria are native versus denaturing conditions and agarose versus polyacrylamide gel matrix Generally recovery of RNA from agarose gels is more efficient and convenient than from polyacrylamide
16. contaminating RNase The RNaid Kit contains RNA Wash Concentrate 1007 203 This component needs to be prepared before use in the protocol Add 120 ml of 100 Ethanol to the bottle of RNA Wash Concentrate and mix well before use 4 Storage The RNaid Kit is shipped at ambient temperature Recommended storage is at ambient temperature 5 Safety Precautions The RNaid Kit contains components RNA Binding Salt and 10 Acetic Acid that when in contact with human tissue may cause minor irritation Wear personal protective 6 www mpbio com equipment to prevent contact with the skin or mucus membranes gloves lab coat and eye protection Consult the Material Safety Data Sheet at www mpbio com for additional details 6 Protocol 6 1 Isolation of RNA from Solution from Agarose or Polyacrylamide Gels Containing up to 6M Urea Note Reagents for agarose urea gels are not included with the kit For an agarose gel in 0 5X TAE buffer containing up to 6M urea prepare two solutions Solution A 1 Dissolve 8M urea in water by heating to 60 C 2 Cool to room temperature and adjust pH to 3 8 with solid citric acid 3 Add approximately 0 8g citric acid 100 ml 8M urea use free acid not sodium salt Solution B 1 Prepare a 4X agarose solution in 2X TAE buffer pH 6 0 2 Melt agarose completely by boiling 3 Mix Solution A with 1 4 volume melted Solution B and cast gel The final concentration is 6M urea and 0 5x TAE at t
17. he desired agarose concentration The gel will solidify within 30 to 60 minutes at 4 C An agarose concentration of less than 1 may take overnight at 4 C to solidify The gel will remain clear upon solidification 4 Load sample and run at 4 C Heat denature RNA sample before loading by incubation at 60 C for 10 minutes in the presence of 50 formamide or at 80 C for 10 minutes without formamide Isolation of RNA from Solution 1 Add 3 volumes of RNA Binding Salt and mix well 2 Continue with Step 3 below Isolation of RNA from Agarose 1 Excise desired RNA band from ethidium bromide stained gel and determine approximate volume by its weight 1 mg 1 ul volume Place gel slice in microcentrifuge tube www mpbio com 7 2 RNaid Kit Add 3 volumes of RNA Binding Salt i e to 0 1 g gel slice add 0 3 ml of RNA Binding Salt Mix and incubate at room temperature for 10 minutes to dissolve agarose Alternatively place tube in 45 55 C water bath to dissolve agarose more rapidly Continue with Step 3 below Isolation of RNA from Polyacrylamide 1 Excise band from ethidium bromide stained gel and determine approximate volume by weight Place into microcentrifuge vial If the gel concentration is 10 or greater then crush or cut the gel into small pieces Add 3 volumes of RNA Binding Salt Soak for 20 minutes at 60 C Remove liquid with small bore pipet tip avoiding gel pieces and transfer to new vial Add 2 ul of 10
18. icals Tel 02 466 00 00 ls cis ae Fax 02 466 26 42 Canada Poland MP Biomedicals Canada MP Biomedicals Poland Tel 888 362 5487 Tel 48 22 659 58 95 Fax 514 935 7541 Fax 48 22 658 45 05 www mpbio com MP Biomedicals 29525 Fountain Parkway Solon OH 44139 tel 1 800 854 0530 fax 1 800 334 6999
19. ing tube to room temperature to prevent precipitation Volume is approximately 110 ul at this point www mpbio com 11 RNaid Kit Purification of RNA with RNAMATRIX T s s 12 Add 3 volumes of RNA Binding Salt and mix Do not precipitate or gel purify RNA prior to adding RNA Binding Salt and RNAMATRIX Pre purifying the hot RNA can lead to very tight binding to the RNAMATRIX and will be difficult to elute Estimate the amount of transcripts and add 1 2 ul of RNAMATRIX per ug of RNA add a minimum of 5 ul of RNAMATRIX Mix well and incubate at room temperature for 5 minutes with occasional mixing to allow adsorption of RNA to the matrix Spin for 1 minute in microcentrifuge at maximum speed and remove and discard supernatant which contains most of the unincorporated label Follow precautions and regulations for handling and disposing of radioactive materials as specified in Radioactive Materials License Wash pellet two times with 500 ul RNA Wash Solution remember to add the correct amount of ethanol before first use see section 3 for instructions and resuspend pellet completely by mixing with pipet tip Spin for 1 minute in microcentrifuge at maximum speed and remove supernatant Remove residual traces of liquid and elute RNA with appropriate volume of DEPC Treated Water by carefully resuspending pellet and incubating at 50 C for 3 minutes Spin for 1 minute and remove supernatant containing la
20. m negative bacteria in 40 seconds e Protein extracts are ready for immediate electrophoresis or purification e Ideal for optimizing induction conditions FastProtein Red Matrix Cat N 6550 600 50 preps Cat N 6550 700 100 preps e Release of proteins from yeast cells and fungi in 40 seconds e Protein extracts are ready for immediate electrophoresis or purification e Ideal for optimizing induction conditions www mpbio com Instruction Manual RNaid Kit Revision 1007 200 100CT Worldwide Ordering and Technical Support United States of America France Serbia Worldwide Headquarters MP Biomedicals France MP Global d o o Tel 1 440 337 1200 Tel 03 88 67 54 25 Tel 381 11 2622 945 Toll Free Tel 800 854 0530 Fax 03 88 67 19 45 Fax 381 11 2623 373 Fax 1 440 337 1180 Toll Free Fax 800 334 6999 Germany Singapore MP Biomedicals MP Biomedicals Singapore Europe Phone 0800 426 67337 Tel 65 6775 0008 Toll Free Phone Fax 0800 629 67337 Fax 65 6775 4536 00800 7777 9999 Toll Free Fax 00800 6666 8888 Switzerland Japan MP Biomedicals Switzerland Australia MP Bio Japan K K Tel 061 271 0007 MP Biomedicals Australasia Tel 03 3808 2102 Fax 061 271 0084 Pty Ltd Toll Free Tel 0120 788 020 Tel 61 2 9838 7422 Fax 03 3808 2401 United Kinad Fax 61 2 9838 7390 nee Neg MP Biomedicals UK Tel 0800 282 474 i The Netherlands Fax 0800 614 735 Belgium MP Biomedicals Netherlands MP Biomed
21. st the pH of the RNA Binding Salt to pH 5 0 by adding 2 ul of 10 Acetic Acid included with kit per 1 ml of RNA Binding Salt and add 3 volumes to the gel slice i e to 0 1 g gel slice add 0 3 ml Binding Salt Acetic Acid mixture The lower pH will optimize the binding efficiency of RNA to the RNAMATRIX Incubate at 37 C for approximately 10 minutes with occasional mixing to melt agarose 3 When gel is completely melted place vial at room temperature and add 1 2 ul of RNAMATRIX per ug of RNA add a minimum of 5 ul of RNAMATRIX Mix well and allow RNA to adsorb to the matrix for 10 minutes at room temperature with periodic mixing www mpbio com 9 RNaid Kit Centrifuge for 1 minute in microcentrifuge at maximum speed to pellet the RNA RNAMATRIX complex Remove supernatant to new tube and save for possible re adsorption Briefly spin again to collect remaining liquid in bottom of the tube Remove all traces of liquid with a small bore pipet tip Resuspend pellet in same amount of RNA Binding Salt as in Step 2 to wash pellet and help remove remaining traces of agarose and formaldehyde Mix thoroughly with pipet tip Spin for 1 minute and remove supernatant Pulse spin and remove traces of liquid with small bore pipet tip Resuspend pellet in 500 ul RNA Wash solution remember to add ethanol before first use see Section 3 above by mixing with pipet tip Spin for 1 minute and remove supernatant Repeat washing Step 6 at least 1
22. than 30 minutes e Variety of soil and environmental sample types e No hazardous organic reagents required e SPIN filters streamline silica handling 18 www mpbio com FastRNA Pro Blue Kit Cat N 6025 050 50 preps e For use with gram positive and gram negative bacteria e Lyse up to 10 cells per 2ml tube e Lysis and isolation with single phase organic solution in less than 90 minutes FastRNA Pro Red Kit Cat N 6035 050 50 preps e For use with yeast cells and fungal tissue e Lyse up to 10 cells per 2ml tube e Lysis and isolation with single phase organic solution in less than 90 minutes FastRNA Pro Green Kit Cat N 6045 050 50 preps e For use with all plant and animal samples e Lyse 50 100 mg tissue per 2ml tube e Lysis and isolation with single phase organic solution in less than 90 minutes FastRNA Pro Soil Direct Kit and FastRNA Pro Soil Indirect Kit Cat N 6070 050 Cat N 6075 050 respectively 50 preps e Isolate RNA from soil samples direct kit and washed soil indirect kit in less than 2 hours e Variety of soil and environmental sample types e RNA protected during and after processing e Humic acids reduced to allow uninhibited RT PCR e Includes additional reagents for even further purification if necessary e SPIN filters streamline silica handling FastProtein Blue Matrix Cat N 6550 400 50 preps Cat N 6550 500 100 preps e Release of proteins from gram positive and gra
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