FastRNA® Pro Blue Kit
Contents
1. Any Questions Call Technical Support at 800 424 6101 7 FastRNA Pro Blue F 8 Add 300 ul of chloroform NO isoamyl alcohol Vortex 10 seconds and then incubate 5 minutes at room temperature 9 Centrifuge the tubes at a minimum of 12 000 x g for 5 minutes at 4 C 10 Transfer the upper phase without disturbing the interphase to a new microcentrifuge tube 11 Add 500 ul of cold absolute ethanol invert 5X to mix and store at 20 C for at least 30 minutes 12 Centrifuge at a minimum of 12 000 x g for 15 minutes at 4 C and remove the supernatant 13 Wash the pellet with 500 yl of cold 75 ethanol made with DEPC H 0 14 Remove the ethanol air dry 5 minutes at room temperature DO NOT completely dry the RNA and resuspend the RNA in 100 pl of DEPC H 0 15 Incubate 5 minutes at room temperature 16 Determine the RNA concentration a Dilute 5 ul of RNA into 495 pl of DEPC H20 b Read the ODs gq using DEPC H90 as a blank c Calculate the sample ug RNA per ml using the formula 0D gt gp 40 ug mI per OD 100 dilution factor ug RNA per ml 17 Aliquot and store the RNA solution at 70 C 18 RNA integrity can be analyzed visually using denaturing or non denaturing 1 2 agarose gel elec trophoresis See Figure 1 2 3 6 Detailed Protocol 1 Dilute 1 ml of an overnight bacterial culture into 14 ml of fresh media in a sterile 50 ml tube or 250 ml flask 2 Incubate for 4 6 hours at 37 C with shaking2. Bacterial strain variability may result in unwanted protein and mucopolysaccharide carryover into the aque ous solution following chloroform extraction While this may not compromise downstream applications the user may adapt the protocol to include an additional chloroform isoamyl alcohol may be included with the chloroform CHCI3 IAA 24 1 v v extraction after Step 10 Quick Protocol for Experienced Users or in Step 12 Detailed Procedure to reduce the potential carryover 6 visit us on the web at www gbiogene com RNA Pro Blue Kit A single 40 second run at a speed setting of 6 0 in the FastPrep Instrument is sufficient to lyse a bacter ial sample If the user determines that additional processing steps in the FastPrep Instrument are required to homogenize a sample it is recommended that the sample be incubated on ice in the sample tube for at least 2 minutes between successive FastPrep Instrument homogenizations to prevent sample heating and possible RNA degradation The FastRNA Pro Blue Kit is designed to selectively purify total cellular RNA from DNA and protein Experiments have indicated the RNA is sufficiently pure for use in RT PCR and northern analysis however it is recommended the user incorporate DNase treatment of the RNA prior to use in applications where absolute control of DNA contamination is essential Use DNase at the concentration recommended by the manufacturer and incubate at 37 C for 30 minutes
3. soi ot oe ae shale Gece tie case cae ceed 11 7 1 Degraded RNA or Lower than Expected Yields een 11 7 2 No Pellet after Ethanol Precipitation ccccccecsccscsecscsecscscscecscessssesssesseeseees 12 T gt Genomic DNA Contamination cios dicen dintel a 12 7 4 Mucopolysaccharide Carbohydrate Contamination 12 7 5 Lithium Chloride Precipitation cccccccccscsscscsecsssecsesscsecscsscscsecsssessssesssesseesseees 12 8 Recommended Reference Format for Publication 13 9 References u 0 2 a a ee 13 10 7 Related Products meer 13 11 Product Use Limitation amp Warranty 0 0 0 cece eee e eee eee eee 14 4 visit us on the web at www qbiogene com tRNA Pro Blue Kit 1 Introduction to the FastRNA Pro Blue Kit and the FastPrep Instrument The FastRNA Pro Blue Kit is a single reagent extraction method designed to quickly and efficiently isolate total cellular RNA from gram positive and gram negative bacteria The RNApro Solution included in the kit is designed to efficiently inactivate cellular RNases during cell lysis to prevent RNA degradation During use the RNApro Solution is mixed with the bacterial sample in a tube containing a specifically selected lysing matrix The tube is then processed in the FastPrep Instrument for 40 seconds to release the total cellular RNA DNA and proteins Following the FastPrep homogenization the RNA is purified and iso
4. 80 C For longer term storage RNA samples may be stored at 20 C as ethanol precipitates When stored as an ethanol precipitate the RNA must be precipitated and resuspended in aqueous solu ion prior to use NOTE RNA does not evenly distribute in ethanol and can lead to inconsistent RNA amounts between samples when equal volumes are pipetted Vortex the RNA ethanol solution to dis perse the RNA prior to removing the sample In situations where precise amounts of RNA are required it is best to precipitate the total amount or excess of RNA required resuspend the RNA in DEPC H20 and measure the concentration by OD gg before proceeding ncubate 5 minutes at room temperature to facilitate RNA resuspension Determine the RNA concentration a Dilute 5 ul of the purified RNA into 495 pl of DEPC H20 b Read the ODs gq using DEPC H90 as a blank c Calculate the sample ug RNA per ml using the formula OD3g0 40 ug mI per OD 100 dilution factor ug RNA per ml Spectrophotometer accuracy is greatest between 0 2 and 0 8 If the OD reading is below the range add more RNA sample e g 20 pl RNA 480 pl DEPC H 0 or concentrate the RNA by precipitation and resuspension into a smaller volume If the OD reading is above the recommended spectropho tometer range use less RNA for the OD determination Aliquot and store the RNA solution at 70 C The RNA integrity can be determined by analyzing a portion of the RNA sample using gel e
5. The DNase is inactivated by incubation at 75 C for 5 minutes or by addition of EDTA to 25 mM followed by phenol chloroform extraction and precipitation 2 3 4 Safety Precautions The RNApro Solution contains components that when in contact with human tissue or during inhalation may cause irritation or burning Wear personal protective equipment to prevent skin contact e g gloves lab coat and eye protection and prevent inhalation of reagent vapors and consumption of liquid during use Consult the enclosed Material Safety Data Sheet for additional details 5 Quick Protocol for Experienced Users 1 Dilute 1 ml of an overnight bacterial culture into 14 ml of fresh media in a sterile 50 ml tube and incubate for 4 6 hours to reach an ODgoo 0 9 1 0 2 Remove 10 ml of the culture to a 15 ml conical tube and pellet the cells by centrifugation Decant the supernatant and add 1 ml of RNApro solution to the tube and resuspend the cells by pipetting or vortexing 3 Transfer 1 ml of the bacterial solution mixture to a blue cap tube containing Lysing Matrix B provided in the kit 4 Process the tube in the FastPrep Instrument for 40 seconds at a setting of 6 0 5 Remove and centrifuge the tube at a minimum of 12 000 x g for 5 minutes at 4 C 6 Transfer the liquid 750 ul to a new microcentrifuge tube Avoid transferring the debris pellet and lysing matrix 7 Incubate the transferred sample 5 minutes at room temperature
6. at 150 200 rpm to reach an ODgoo 0 9 1 0 Note 1 0 ODgoo for Escherichia coli is 1 X 109 cells per milliliter 3 Remove 10 ml of the culture to a 15 ml conical tube and pellet the cells by centrifugation at 2 800 rpm x 1 500 g for 15 minutes at 4 C e g Beckman Model TJ 6 Centrifuge l 92 Swinging Bucket Rotor for 10 minutes 8 visit us on the web at www gbiogene com tRNA Pro Blue Kit 4 Decant the supernatant and add 1 ml of RNApro Solution to the tube Completely resuspend the cells by pipetting or vortexing 5 Transfer 1 ml of the resuspended cells to a blue cap tube containing Lysing Matrix B provided in the kit Securely close the cap to prevent leakage in the next step NOTE The calculated volumes will pro vide adequate airspace in the matrix tube to prevent sample leakage and or tube failure DO NOT over fill the matrix tube To process a greater number of cells use a second matrix tube 6 Process the sample tube in the FastPrep Instrument for 40 seconds at a setting of 6 0 7 Remove the sample tube and centrifuge at a minimum of 12 000 x g for 5 minutes at 4 C or room temperature 8 Transfer the liquid 750 ul to a new microcentrifuge tube Avoid transferring the debris pellet and lysing matrix 9 Incubate the transferred sample 5 minutes at room temperature to increase RNA yield 10 Add 300 ul of chloroform NO isoamyl alcohol Vortex 10 seconds 11 Incubate 5 minutes at room temperatu
7. 35 050 FastRNA Pro Green Kit Plant amp Animal 50 preps 6045 050 FastRNA Pro Soil Kit 50 preps 6070 050 FastDNA Kit 100 preps 6540 400 FastDNA SPIN Kit for Soil 50 preps 6560 200 FastPROTEIN Blue Matrix 50 preps 6550 400 FastPROTEIN Red Matrix 50 preps 6550 600 RNase Erase 500 ml 2440 204 Lysing Matrix B 50 x 2ml tubes 6911 050 BGFNE alkaline agarose gel loading dye 1 ml 2339 104 BBXFE denaturing RNA gel loading dye 1 ml 2343 104 BBG general purpose neutral gel RNA and DNA loading dye 1 ml 2327 104 BBG general purpose neutral gel RNA and DNA loading dye 1 ml 2327 104 Any Questions Call Technical Support at 800 424 6101 13 FastRNA Pro Blue K 11 Product Use Limitation amp Warranty Unless otherwise indicated this product is for research use only Purchase of Qbiogene Inc products does not grant rights to reproduce modify or repackage the products or any derivative thereof to third parties Qbiogene Inc 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 Qbiogene Inc hereunder shall be limited to at our dis cretion no replacement or compensation product credits refund of the purchase price of or the replace ment of materials that do not meet our specification By acceptance of the product Buyer ind
8. FastRNA Pro Blue Kit Rapid Isolation of Total RNA from Gram Positive and Gram Negative Bacteria Using the FastPrep Instrument Revision 6025 050 3F24 RNA Pro Blue Kit FastRNA Pro Blue Kit Rapid Isolation of Total RNA from Gram Positive and Gram Negative Bacteria Using the FastPrep Instrument Application Manual Revision 6025 050 3F24 Catalog 6025 050 50 Samples Storage temperature Refrigerated or ambient temperature 4 C or 15 30 C DO NOT expose RNApro Solution to light for extended periods of time Store in the original bottle in the closed kit box Note An empty space in the box insert has been provided for convenient storage and access to the RNApro Solution when it has been removed from the safety shipping container Any Questions Call Technical Support at 800 424 6101 3 FastRNA Pro Blue H TABLE OF CONTENTS 1 Introduction to the FastRNA Pro Blue Kit and the FastPrep Instrument 5 2 Kit Components and User Supplied Materials 5 2 1 FastRNA Pro Blue Kit COMPONENHS ccccccccscssesscsscssssscsscsecsesessessessessessesseseeses 5 2 2 User Supplied Materials znr 6 3 Important Considerations before Use 2222222 eee e cece eeeeeeee 6 4 safety Precautions rare ee ee en 7 5 Quick Protocol for Experienced Users c cece eee ee cece enna 7 6 Detailed Protocola 4 2 2u u22 ar ER LG 8 7 Troubleshooting
9. and integrity RNA Bacterial cells in stationary phase growing in oxygen or nutrient limiting conditions stored for extended duration at room temperature or refrigerated for extended periods will contribute to reduced RNA yield and integrity Any Questions Call Technical Support at 800 424 6101 i i FastRNA Pro Blue RNApro Solution can permeate samples and will protect bacterial RNA from degradation for at least 24 hours before it is processed in the FastPrep Instrument Artifactual RNA degradation may occasionally occur during gel electrophoresis due to a gel that was not RNase free running the gel at too high voltage or from using depleted running buffer Rerun the samples with a known intact RNA sample using freshly prepared reagents RNA degradation may occur due to RNase contamination introduced into the DEPC H 0 following use If contamination is suspected prepare fresh DEPC H 0 in an RNase free container 2 3 RNApro Solution contains RNase inactivating components and will not support active RNase contamination 7 2 No Pellet after Ethanol Precipitation The purified RNA may not appear as a pellet but may instead adhere to the side of the tube The RNA may not be visible and it MAY APPEAR THAT RNA HAS NOT BEEN PURIFIED COMPLETE THE RNA PURIFICATION per the instructions provided and confirm the RNA concentration by ODsgq and integrity by gel elec trophoresis RNA adhering to the tube wall will not affect i
10. emnifies and holds Qbiogene Inc 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 Qbiogene Inc 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 FastRNA FastDNA FastPrep and BIO 101 Systems are registered trademarks of Qbiogene Inc RNApro is a trademark of Qbiogene Inc 14 visit us on the web at www qbiogene com
11. it procedure Following ethanol precipitation of the RNA and resuspension in 100 ul 12 visit us on the web at www gbiogene com StRNA Pro Blue Kit DEPC H 0 add lithium chloride to a final concentration of 2 3 M e g 0 2 volumes 20 ul RNase free 8 M lithium chloride Add 2 5 volumes RNase free absolute ethanol 250 ul Mix the solution and store on ice at least 2 hours Centrifuge for 15 minutes at a minimum of 12 000 rpm at 4 C Remove the supernatant and wash the pellet with 75 cold RNase free ethanol The ethanol wash step is critical to prevent LiCl inhibition of cell free translation and in vitro transcription Air dry and resuspend the RNA in 100 pl DEPC H 0 8 Recommended Reference Format for Publications Total RNA was isolated from mg of cells using the FastRNA Pro Blue Kit Qbiogene Inc CA and FastPrep Instrument Qbiogene Inc CA for seconds at a speed setting of 9 References 1 U S Patent 5 567 050 Zbloninsky et al Apparatus and method for rapidly oscillating specimen vessels 2 Molecular Cloning Sambrook and Russell Cold Spring Harbor Laboratory Press 3rd Edition 2001 3 Current Protocols in Molecular Biology John Wiley amp Sons Inc 2002 www currentprotocols com 10 Related Products Description Size Catalog FastPrep FP100A Instrument 100V 6001 100 FastPrep FP120A Instrument 120V 6001 120 FastPrep FP220A Instrument 220V 6001 220 FastRNA Pro Red Kit Yeast 50 preps 60
12. l RNase free microcentrifuge tubes Agarose Gel loading dye and RNA size marker 3 Important Considerations before Use The presence or introduction of RNase during the procedure may result in sample degradation It is strongly recommended that the user minimize the potential for RNase contamination by using gloves throughout the procedure using DEPC H20 and by treating pipettmen work area gel box and gel comb with RNase Erase Additional RNA handling methods and precautions may be found in references 2 and 3 The volume after the addition of RNApro Solution to the sample has been calculated to maintain a suffi cient air space in the sample tube during FastPrep Instrument processing Sample loss or tube failure may result from overfilling the matrix tube The matrix tube caps must be secure but not over tightened to pre vent sample leakage If the sample is too large for processing in a single tube divide the sample and process using multiple tubes Confirm the sample tubes spin freely and will not scrape the microcentrifuge wall during centrifugation The use of other manufactured tubes in the FastPrep Instrument is not recommended and may result in sample loss or FastPrep Instrument failure Add the RNApro Solution to the sample as soon as possible to initiate RNase inhibition Samples both FastPrep Instrument homogenized and non homogenized are stable in RNApro Solution overnight at room temperature or 4 C
13. lated by chloroform extraction and ethanol precipitation The purified RNA is ready for downstream applications including RT PCR and northern analysis The average RNA yield from 1010 bacteria is greater than 50 ug The FastPrep Instrument is a high speed benchtop device that uses a proprietary vertical angular motion 1 to produce sample homogenization by simultaneous matrix impaction from multiple directions The FastPrep Instrument provides an extremely quick and highly reproducible homogenization that surpasses traditional lysis methods using enzyme digestion sonication blending douncing and vortexing When used with FastPrep kits the FastPrep Instrument permits the release and purification of intact DNA RNA and proteins from virtually any source including bacteria yeast and fungi spores plant seeds and leaves ani mal tissue organs and blood etc 2 Kit Components and User Supplied Materials 2 1 FastRNA Pro Blue Kit Components Product Description Qty RNApro Solution 1 x 55 ml bottle DEPC H 0 1x 15 ml bottle Lysing Matrix B 50 x 2 ml tubes Short protocol 1 each User manual 1 each MSDS 1 each Certificate of Analysis 1 each Any Questions Call Technical Support at 800 424 6101 5 FastRNA Pro Blue 2 2 User Supplied Materials FastPrep Instrument Cat 6001 100 120 or 220 Microcentrifuge Pipettmen RNase Erase Cat 2440 204 recommended Chloroform 100 ethanol 75 ethanol 1 5 or 2 0 m
14. lectrophore sis Add 1 ug RNA in 9 pl DEPC H 0 heat to 65 C for 5 minutes add gel loading buffer see Related Products and load the sample on a 1 2 agarose gel containing 2 2M formaldehyde in MOPS buffer The sample is run at 80 volts for 30 minutes 2 3 Ethidium bromide may be added to the dena tured RNA sample at a final concentration of 10 ug per milliliter prior to gel loading or the gel may be ethidium bromide stained and destained following electrophoresis and visualized under UV light The quality of the RNA is determined by the appearance of the large 23S and small 16S ribosomal RNAs as sharp distinct bands of 2 9 and 1 5 kb Heterogeneous sized messenger RNA may appear as dif fuse ethidium staining between and below the ribosomal bands Small RNA species such as tRNA and 5S RNA may be present in varying amounts at the dye front visit us on the web at www gbiogene com FastRNA Pro Blue Kit Figure 1 Bacterial total RNA extracted with the FastRNA Pro Blue Kit Approximately 2 of the total RNA isolated from 1010 bacterial cells was loaded on to a 1 2 denaturing agarose gel 1XMOPS Lane 1 Salmonella typhimurium Lane 2 Pseudomonas stuartii Lane 3 Escherichia coli Lane 4 Bacillus subtilis Lane 5 0 24 9 5kb RNA Ladder 7 Troubleshooting 7 1 Degraded RNA or Lower than Expected RNA Yields RNA purified using the FastRNA Pro Blue Kit and analyzed by denaturing or non denaturing agarose gel electrophore
15. re to permit nucleoprotein dissociation and increase RNA purity 12 Centrifuge the tubes at a minimum of 12 000 x g for 5 minutes at 4 C Samples containing large amounts of cellular mucopolysaccharides can be re extracted with chloroform isoamyl alcohol may be included with the chloroform CHCI3 IAA 24 1 v v to increase RNA purity Alternatively a lithium chlo ride precipitation may be used see the Troubleshooting section and references 3 4 13 Transfer the upper phase to a new microcentrifuge tube without disturbing the interphase If a portion of the interphase is transferred repeat the centrifugation with the upper phase and transfer the new upper phase to a clean microcentrifuge tube 14 Add 500 ul of cold absolute ethanol to the sample invert 5X to mix and store at 20 C for at least 30 minutes 15 Centrifuge at a minimum of 12 000 x g for 15 minutes at 4 C and remove the supernatant The RNA will appear as a white pellet in the tube If the pellet is floating the sample may be recentrifuged to place the pellet at the tube bottom 16 Wash the pellet with 500 ul of cold 75 ethanol made with DEPC H 0 Any Questions Call Technical Support at 800 424 6101 9 FastRNA Pro Blue 17 18 19 20 21 Remove the ethanol air dry 5 minutes at room temperature DO NOT completely dry the RNA and resuspend the RNA in 100 ul of DEPC H20 for short term storage RNA is generally stable for up to a year at
16. sis will appear as 2 distinct ribosomal RNA rRNA bands of approximately equal fluorescent intensity using ethidium bromide staining The rRNA bands will appear in the area between 2000 and 1000 nucleotides Messenger RNA mRNA which typically represents approximately less than 1 of the total cel lular RNA and is heterogeneous length will not be visible as distinct bands rRNA is used as a marker to assess sample RNA degradation Degraded RNA or mRNA may appear as unequal fluorescent intensity between bands a single band may be completely lacking or a heterogeneous fluorescent smear may appear below the rRNA bands or throughout the gel lane Recommended precautions include cleaning all instruments and work area with RNase Erase Qbiogene Catalog 2440 204 prior to use Use disposable sterile plastic containers when possible Glassware should be thoroughly cleaned rinsed with DEPC H20 and baked at 250 C for 4 hours to remove RNase Sterile plugged micropipettes are recommended see 2 3 for additional suggestions Certain bacterial strains may contain elevated RNase levels Reduce the exposure time to RNase by adding RNApro Solution to each sample as soon as possible following sample harvest Process fewer samples to shorten the time before complete cellular lysis and exposure to the RNase inactivating activity of RNApro Solution Bacteria in log phase growth with maximal aeration and nutrients provide the highest yield
17. ts purity size or use in subsequent applications The RNA pellet may not be firmly attached to the side of the tube and may be observed floating in the solu tion or at the solution surface Recentrifuge the sample in the same tube and exercise caution to not lose the pellet when removing the supernatant Confirm enough sample was used to isolate RNA 1 0 OD for Escherichia coli 1x10 cells 7 3 Genomic DNA Contamination Genomic DNA contamination will appear as a high molecular weight smear on a denaturing gel or as ethidium bromide stained material in the gel loading well In the event genomic DNA contamination occurs treat sam ple with DNase according to the manufacturer s instructions 7 4 Mucopolysaccharide Carbohydrate Contamination Samples containing large amounts of cellular mucopolysaccharides can be re extracted after the initial chlo roform extraction with a second chloroform extraction Isoamyl alcohol may be included with the chloroform CHCI3 IAA 24 1 v v to increase RNA purity Refer also to Lithium Chloride Precipitation in the Troubleshooting section 7 5 Lithium Chloride Precipitation Lithium chloride LiCl may be used to precipitate RNA while excluding carbohydrate DNA and proteins including transcription inhibitors Lithium chloride has historically been used to precipitate RNA greater than 300 nucleotides from tRNA and 5S RNA Lithium chloride precipitation may be incorporated into the FastRNA Pro Blue K
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