FastDNA® SPIN Kit for Feces
Contents
1. 6 3 3 Recovery of DNA from Dry Samples 7 4 Safety Precautions ccccccsssccsesssssesssessscesssseeessseessseesesenevees i SE OG OO PME RR T REN 7 6 Troubleshooting eeeeeeen nn 9 6 1 Humic Acid contamination esssesssss 9 7 Example Data DNA Isolation from Animal Stool samples and Gel Electrophoresis eeeeee 9 8 Recommended Reference Format for Publication 10 SERIES IE NT 10 10 Related PEOULIBIS eiae ino sumi e pacti fbi idv isan dni In PdT PU TUR Er RU 10 11 Product Use Limitation amp Warranty 11 www mpbio com 1 Introduction to the FastDNA SPIN Kit for Feces and the FastPrep Instruments The FastDNA SPIN Kit for Feces quickly and efficiently isolates PCR ready genomic DNA from fresh or frozen human and animal stool samples Designed for use with the FastPrep Instruments from MP Biomedicals host cells as well as bacteria fungi viruses protists and other cells present in fecal samples are easily lysed within 40 seconds These benchtop devices use a unique optimized motion to homogenize samples by multidirectional simultaneous impaction with lysing matrix particles FastPrep Instruments provide an extremely quick efficient and highly reproducible homogenization that surpasses traditional extraction method2. 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 than 30 minutes e Variety of soil and environmental sample types e No hazardous organic reagents required e SPIN filters streamline silica handling 12 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 sampl
3. 6570 200 10JUN J 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 hited naigepm MP Biomedicals UK Tel 0800 282 474 p The Netherlands Fax 0800 614 735 Belgium A MP Biomedicals Netherlands MP Biomedicals Tel 02 466 00 00 ira dei 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 e 29525 Fountain Parkway e Solon OH 44139 e tel 1 800 854 0530 fax 1 800 334 6999
4. 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 Biomedicals 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 FastDNAS FastRNAS FastPrep QBiogene and BIO 1019 Systems are registered trademarks of MP Biomedicals LLC www mpbio com 11 FastDNA Spin Kit for Feces 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 PROTEIN FastPrep Kits RNA FastDNA Kit and FastDNA Spin Kit
5. 0 15 seconds NOTE See section 3 3 for other important guidelines 2 Centrifuge samples at 14 000 x g for 5 minutes and decant supernatant 3 Add 978 ul Sodium Phosphate Buffer and 122 ul MT Buffer Shake vigorously or vortex briefly to mix 4 Homogenize samples in the FastPrep 24 instrument at setting 6 0m s for 40 seconds 5 Centrifuge samples at 14 000 x g for 5 minutes NOTE Extending centrifugation to 15 minutes can enhance elimination of excessive debris from large samples or from cells with complex walls 6 Transfer the supernatant to a clean 2 0 ml centrifuge tube www mpbio com 7 FastDNA Spin Kit for Feces 7 Add 250 ul of PPS solution shake vigorously to mix and incubate at 4 C for 10 minutes Do not vortex Centrifuge samples at 14 000 x g for 2 minutes 8 While samples are centrifuging add 1 ml of Binding Matrix Solution to a clean 15 ml conical tube not supplied 9 Transfer supernatant to the Binding Matrix Solution in the 15 ml conical Shake gently by hand to mix then place on a shaker rocker for 3 5 minutes 10 Centrifuge samples at 14 000 x g for 2 minutes Decant the supernatant 11 Wash the binding mixture pellet by gently resuspending with 1 ml Wash Buffer 1 12 The following step will require two spins First transfer approximately 600 ul of the binding mixture to a SPIN Filter tube and centrifuge at 14 000 x g for 1 minute Empty the catch tube and add the remaining binding mixtur
6. IN Kit for Soil 50 preps 6560 200 FastDNA 50ml SPIN Kit for Soil 10 preps 6570 200 FastRNA Pro Soil Direct Kit 50 preps 6070 050 FastRNA Pro Soil Indirect Kit 50 preps 6075 050 10 www mpbio com Description Size Catalog FastRNA Pro Red Kit Yeast amp Fungus 50 preps 6035 050 FastRNA Pro Green Kit Plant amp Animal 50 preps 6045 050 FastRNA Pro Blue Kit Bacteria 50 preps 6025 050 FastProtein Blue Matrix 50 preps 6550 400 FastProtein Red Matrix 50 preps 6550 600 Lysing Matrix E 50 x 2 ml tubes 6914 050 Lysing Matrix E 100 x 2 ml tubes 6914 100 Lysing Matrix E 500 x 2 ml tubes 6914 500 11 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
7. Instruction Manual E FastDNA SPIN Kit for Feces Rapid Isolation of Genomic DNA from Human and Animal Stool Samples Using the FastPrep System One Call 50 P One Source T A World of otorage Ambient temperature 15 30 C B I olech n ol Og y Catalog 6570 200 Reagents Revision 65 0 200 10JUN 2 m www mpbio com MP Biomedicals e 29525 Fountain Parkway e Solon OH 44139 e tel 1 800 854 0530 fax 1 800 334 6999 FastDNA Spin Kit for Feces TM MP Biomedicals Our family of companies includes w BIO 101 Systems www mpbio com FastDNA SPIN Kit for Feces Rapid Isolation of Genomic DNA from Human and Animal Stool Samples Using the FastPrep System 50 Preps storage Ambient temperature 15 30 C Catalog 6570 200 Revision 65 0 200 10JUN www mpbio com FastDNA Spin Kit for Feces TABLE OF CONTENTS 1 Introduction to the FastDNA SPIN Kit for Feces and the FastPrep Instruments enne 5 2 Kit Components and User Supplied Materials 5 2 1 FastDNA SPIN Kit for Feces Components 5 2 2 User Supplied Materials eeeee 6 3 Important Considerations Before Use 6 3 1 IPreparation of Wash Buffer 2 Solution 6 3 2 Sample Lysis with the FastPrep Instrument
8. als FastPrep Instrument see Section 9 Microcentrifuge that can freely spin 2 0 ml tubes Microcentrifuge tubes 2 0 ml and 1 5 ml sterile 15 ml tubes for DNA binding Rotator or low speed vortex 100 Ethanol 3 Important Considerations Before Use 3 1 Preparation of Wash Buffer 2 The FastDNA SPIN Kit for Feces contains Wash Buffer 2 which is a plastic screw top bottle containing 12 ml of a concentrated salt wash solution Before using this buffer add 100 ml of 100 ethanol and mark on the bottle label the date ethanol was added Ensure that the bottle is securely closed to prevent evaporation and store at room temperature 3 2 Sample Lysis with the FastPrep Instrument The fill volume in the lysing matrix tube after the addition of the Sodium Phosphate and MT Buffer Solution to the sample should allow sufficient air space in the sample tube for efficient FastPrep Instrument processing MP Biomedicals recommends using 500 mg of starting material as long as there is between 250 500 ul of empty space in the tube Sample loss or tube failure may result from overfilling the matrix tube The matrix tube caps must be secure but not over tightened to prevent sample leakage If the sample is too large for processing in a single tube divide the sample and process using multiple tubes MP Biomedicals Lysing Matrix particles and tubes have been rigorously tested and validated in the FastPrep Instrument The use of other products w
9. e contains 0 1 mm silica spheres Lysing Matrix B tubes have blue caps and are found in the FastRNA Pro Blue Kit and FastProtein Blue Matrix Lysing Matrix B is used for lysis of gram positive and gram negative bacteria Lysing Matrix C Each impact resistant 2 ml tube contains 1 mm silica spheres Lysing Matrix C tubes have red caps and are found in the FastRNA Pro Red Kit and FastProtein Red Matrix Lysing Matrix C is used for lysis of yeast and fungi Lysing Matrix D Each impact resistant 2 ml tube contains 1 4 mm ceramic spheres Lysing Matrix D tubes have green caps and are found in the FastRNA Pro Green Kit for isolation of total RNA from plants and animals Lysing Matrix E Each impact resistant 2 ml tube contains 1 4 ceramic spheres 0 1 mm silica spheres and one 4 mm glass bead Lysing Matrix E tubes have purple caps and found in the FastDNA SPIN Kit for Soil and the FastRNA Pro Soil Kits www mpbio com Dirty Samples Tiny or Huge Tough Samples Samples No Problem with FastPrep fastprep Automated Lysis part of an MP BIO integrated laboratory solution Sample Prep Higher yields Consistent quality Super fast FastPrep lets you get the most usable DNA RNA and proteins from even your most difficult samples fast and easy every time FREE fastprep DEMO Learn more at www mpSamplePrep com www mpbio com 15 Instruction Manual FastDNA SPIN Kit for Feces Protocol Revision
10. e to the SPIN Filter tube and centrifuge as before Empty the catch tube again 13 Add 500 ul of prepared Wash Buffer 2 to the SPIN Filter tube and gently resuspend using the force of the liquid from the pipette tip to resuspend the pellet Do not vortex NOTE Ensure that ethanol has been added to the Wash Buffer 2 See section 3 1 14 Centrifuge samples at 14 000 x g for 2 minute Discard the flow through 15 Centrifuge the sample again for 2 minutes to extract residual ethanol from the binding matrix and dry the sample 16 Transfer the SPIN Filter bucket to a clean 1 9 ml Catch Tube Add 50 100 ul TES Flick the tube or stir the matrix with a pipette tip to resuspend the pellet Do not vortex 17 Centrifuge samples at 14 000 x g for 2 minutes to elute purified DNA into the clean catch tube Discard the SPIN filter DNA is now ready for PCR and other downstream applications Store at 20 C for extended periods or 4 C until use 8 www mpbio com 6 Troubleshooting 6 1 Humic Acid Contamination some stool samples contain very high levels of humic acids which may co purify with the genomic DNA PCR applications are hindered by the presence of humic acid If the final eluted DNA returns a negative PCR result then humic acid contamination might be the reason The FastDNA SPIN Kit for Feces is designed to eliminate humic acid contamination in feces If humic acid contamination is suspected repeat the protocol from the beginning wi
11. e types e RNA protected during and after processing e Humic acids reduced to allow uninhibited RT PCR e ncludes 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 gram negative bacteria in 40 seconds e Protein extracts are ready for immediate electrophoresis or purification e deal 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 deal for optimizing induction conditions www mpbio com 13 FastDNA Spin Kit for Feces Convenient Lysing Matrix Tubes for Every Need MP Biomedicals guarantees the BEST performance from your FastPrep 24 Instrument when used in combination with 14 FastPrep Lysing Matrix Tubes aS Ps d W Lysing Matrix A Each impact resistant 2 ml tube contains garnet matrix and one 1 4 inch ceramic sphere Extra 1 4 inch ceramic spheres are packaged separately Lysing Matrix A tubes have orange caps and are found in the FastDNA and FastDNA SPIN Kits Lysing Matrix A is used for all sample types except soil for the subsequent isolation of genomic DNA Lysing Matrix B Each impact resistant 2 ml tub
12. ith the FastPrep Instrument is not recommended and may result in sample loss or instrument failure Asingle 40 second run at a speed setting of 6 0 in the FastPrep Instrument is sufficient to lyse almost all samples If the user experimentally determines that additional processing time is required the sample should be incubated on ice in the Lysing Matrix E tube for at least 2 minutes between successive FastPrep Instrument homogenizations to prevent overheating the sample and tube 6 www mpbio com 3 3 Recovery of DNA from Dry Samplest To optimize DNA recovery from extremely dry samples stool samples should be solubilized in Sodium Phosphate Buffer in a separate tube Weigh approximately 500 mg of dry stool and add an equivalent volume of buffer 500 ul Vortex the sample at a low speed to create a homogeneous solubilized stool sample Transfer 500 mg of the solubilized sample to the Lysing Matrix E tube and process as normal 4 Safety Precautions PLS Solution Binding Matrix and Wash Solution 2 contain components that when in contact with human tissue may cause irritation Wear personal protective 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 5 Protocol 1 Ina2 ml Lysing Matrix E tube add 500 mg grams feces 825 ul Sodium Phosphate Buffer and 275 ul of PLS solution Shake to mix then vortex 1
13. s using enzymatic digestion sonication blending douncing and vortexing Samples are placed into 2 0 ml tubes containing Lysing Matrix E a mixture of ceramic and silica particles designed to efficiently lyse all stool organisms Stool samples are pre treated by washing in a Pre Lysis Buffer Homogenization in the FastPrep Instrument with Lysing Matrix E takes place in the presence of MT Buffer and sodium Phosphate Buffer The pre lysing and lysing reagents were carefully developed to eliminate contaminants and to protect and solubilize nucleic acids upon cell lysis These reagents work together to facilitate extraction of genomic DNA with minimal RNA or humic acid contamination Following lysis samples are centrifuged to pellet debris and lysing matrix DNA is purified from the supernatant with a silica based GENECLEAN9 procedure using SPIN filters Eluted DNA is ready for digestion electrophoresis PCR and any other desired application 2 Kit Components and User Supplied Materials 2 1 FastDNA SPIN Kit for Feces Components Lysing Matrix E 50x 2 0 ml tubes PLS Solution 25 ml MT Buffer 8 ml Sodium Phosphate Buffer 100 ml PPS 25 ml Binding Matrix 66 ml Wash Buffer 1 120 ml Wash Buffer 2 12 ml TES 20 ml BBS Gel Loading Dye 200 ul SPIN Filter Tubes 50 each Catch Tubes 50 each User manual 1 each MSDS Online www mpbio com 1 each Certificate of Analysis 1 each www mpbio com 5 FastDNA Spin Kit for Feces 2 2 User Supplied Materi
14. th a fresh stool sample and repeat step 1 once as well as steps 10 amp 11 once each in order T Example Data DNA Isolation from Animal Stool Samples and Gel Electrophoresis DNA from stool samples extracted with the FastDNA Kit for Feces Approximately 200 ng of isolated DNA was loaded on a 1 2 agarose gel 0 5X TAE Lane 1 Lambda Hind Ill marker Lane 2 500 mg bovine stool Lane 3 500 mg equine stool Lane 4 500 mg feline stool Lane 5 500 mg avian stool www mpbio com 9 FastDNA Spin Kit for Feces 8 Recommended Reference Format for Publications DNA was isolated from specific sample using the FastDNA SPIN Kit for Feces and the FastPrep Instrument MP Biomedicals Santa Ana CA 9 References Ando A et al 2005 Appl Envir Microbiol Nov 71 11 7075 82 B lum J et al 2006 Appl Envir Microbiol Feb 72 2 1476 86 Braid MD et al 2003 J Microbiol Methods Mar 52 3 389 93 Dong D et al 2006 J Microbiol Methods Aug 66 2 21 7 22 Kuntz RL et al 2004 Water Res Sep 38 16 3551 7 Layton A et al 2006 Appl Envir Microbiol Jun 72 6 421 4 24 10 Related Products Description Size Catalog FastPrep 24 Instrument 100 230V 6004 500 FastPrep FP100A Instrument 100V 6001 100 FastPrep FP120A Instrument 120V 6001 120 FastPrep FP220A Instrument 220V 6001 220 FastDNA Kit 100 preps 6540 400 FastDNA SPIN Kit 100 preps 6540 600 FastDNA SP
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