FastDNA-96™ Soil Microbe DNA Kit
Contents
1. 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 RNA protected during and after processing Humic acids reduced to allow uninhibited RT PCR e Includes additional reagents for even further purification if necessary SPIN filters streamline silica handling FastProtein Blue Matrix Cat N 6550 400 50 preps Cat N 6550 500 100 preps Release of proteins from gram positive and gram negative bacteria in 40 seconds 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 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 15 7 Application Manual Revision 9696 400 11FEB Worldwide Ordering and Technical Support Russia MP Biomedicals Russia Tel 7 095 995 2844 Fax 7 095 995 2846 gt Serbia and Montenegro MP Global d o o Tel 381 11 2622 945 Fax 381 11 2623 373 Singapore a MP Biomedicals Asia Pacific Pte Ltd Tel2. 65 6775 0008 Fax 65 6775 4536 United Kingdom MP Biomedicals UK Tel 0800 282 4 Fax 0800 614 735 Germany MP Biomedicals Gmbh Phone 0800 426 67337 Fax 0800 629 67337 Italy MP Biomedicals Italy Tel 0800 011 643 Fax 0800 255 220 Japan MP Bio Japan K K Tel 03 3808 2102 Toll Free Tel 0120 788 020 Fax 03 3808 2401 The Netherlands MP Biomedicals Netherlands Tel 0800 0227416 Fax 0800 0227489 Poland MP Biomedicals Poland Tel 48 22 659 58 95 Fax 48 22 658 45 05 United States of America Worldwide Headquarters Tel 1 440 337 1200 Toll Free Tel 800 854 0530 Fax 1 440 337 1180 Toll Free Fax 800 334 6999 Europe Toll Free Phone 00800 7777 9999 Toll Free Fax 00800 6666 8888 Australia MP Biomedicals Australasia Pty Ltd Tel 61 2 9838 7422 Fax 61 2 9838 7390 Canada MP Biomedicals Canada Tel 888 362 5487 Fax 514 935 7541 France MP Biomedicals France Tel 03 88 67 25 Fax 03 88 67 5 www mpbio com MP Biomedicals 29525 Fountain Parkway Solon OH 44139 tel 1 800 854 0530 fax 1 800 334 6999
3. Plate Prep Solution 30 ml Deep Well Plate 2 each MP 96 Binding Plate 2 each MP 96 Inhibitor Removal Plate 2 each Collection Plate 2 each Elution Plate 6 each Foil Plate Cover 4 each User manual 1 each MSDS Online www mpbio com 1 each Certificate of Analysis 1 each 2 2 Storage All FastDNA 96 Soil Microbe DNA Kit reagents are stable at room temperature Storage should be maintained at room temperature The kit reagents are guaranteed for up to one year from the date of purchase of the kit 2 3 User Supplied Materials FastPrep 96 Instrument see Section 10 Centrifuge with a swing bucket style rotor that can spin up to 3 500 5 000 rpm Swing bucket centrifuge microplate adaptors for 96 well plates pair Plate shaker Sterile water 3 Important Considerations Before Use 3 1 Binding Plate Pre Wash Buffer The FastDNA 96 Soil Microbe DNA Kit contains a bottle of Binding Plate Pre Wash Buffer which may form a precipitate over time The precipitate is easily resuspended in solution by gently warming the bottle at 30 37 C for up to 30 minutes with mixing by inversion IMPORTANT Do not microwave this reagent FastDNA 96 Soil M 3 2 MP 96 Inhibitor Removal Plate Preparation Prior to executing the protocol in Section 5 the MP 96 Inhibitor Removal Plate must be prepared for proper use in the assay First mount the MP 96 Inhibitor Removal Plate on top of a supplied Elution Plate Add 150 ul of Inhibitor Pla
4. lyse all soil organisms including historically difficult sources such as eubacterial spores and endospores gram bacteria yeast algae nematodes and fungi while in the presence of a specially formulated lysis solution The FastDNA 96 Soil Microbe DNA Kit isolates microbial DNA from up to 130mg of soil or sludge through a purification process that eliminates completely polyphenols and humic FastDNA 96 Soil Mi acids Organic denaturants or proteinases are not needed with this procedure Alternatively DNA can be isolated from bacterial or fungal broth cultures directly Purified humic free DNA is eluted in an EDTA free DNA elution solution and is ready for downstream applications including digestion electrophoresis and PCR A260 A280 ratios gt 1 8 Yield is typically 5 ug of total DNA eluted in 50 100 ul of elution solution The FastDNA 96 Soil Microbe DNA Kit will recover genomic DNA fragments from 25 kb to 35 kb however some experiments have yielded results from as little as 100 bp up to gt 40 kb Viral and parasitic DNA will also be isolated if these hosts are present in your samples 2 Kit Components Storage and User Supplied Materials 2 1 FastDNA 96 Soil Microbe DNA Kit Components FastDNA 96 Lysing Matrix Rack 0 5 mm Beads 2 x 96 well rack Lysis Buffer 2x 40 ml Soil DNA Binding Solution 150 ml Binding Plate Pre Wash Buffer 50 ml Soil DNA Wash Buffer 100 ml Elution Solution 2x10ml Inhibitor
5. 4 y Application Manual Instruction Manual Rapid High Throughput Isolation of PCR Ready Genomic DNA from Soil Samples using the FastPrep 96 System Catalog 9696 200 2 x 96 Preps Storage Ambient temperature 15 30 C Revision 9696 200 11FEB MP Biomedicals 29525 Fountain Parkway Solon OH 44139 e tel 1 800 854 0530 fax 1 800 334 6999 FastDNA 96 Soil Microbe FastDNA 96 Soil Microbe DNA Kit Rapid High Throughput Isolation of PCR Ready Genomic DNA from Soil Samples using the FastPrep 96 System Catalog 9696 200 2 x 96 Preps Storage Ambient temperature 15 30 C Revision 9696 200 11FEB FastDNA 96 Soil Micre TABLE OF CONTENTS 1 Introduction to the FastDNA 96 Soil Microbe DNA Kit and the FastPrep 96 9 5 2 Kit Components Storage amp User Supplied 131671815 6 2 1 FastDNA 96 Soil Microbe DNA Kit Components ssssssssssses 6 22 50 7 2 3 User Supplied Materials 7 3 Important Considerations Before USe ssssssssscsessssessesssssensessseeseees 7 3 1 Binding Plate Pre Wash Buffer ssssssssscssseccssecsseecsneecsnseeseeesscesees 7 3 2 MP 96 Inhibitor Removal Plate Preparation 8 4 Safety PreCaUtions csscssssecsssecsssecssccssecseecsseecsscessseeesneecsnccesneceseeesseestes 8 8 6 Example Data DNA Isolation from Soil Samples and Gel
6. 7 Recommended Reference Format for Publication 7 10 Product Use Limitation amp 3 1 Introduction to the FastDNA 96 Soil Microbe DNA Kit and the FastPrep 96 Instrument The FastDNA 96 Soil Microbe DNA Kit quickly and efficiently isolates humic free PCR ready genomic DNA from soil samples in approximately 50 minutes Gram bacteria fungi plant and animal tissues algae spores and many other members of a soil population including clay sandy silty peaty chalky and loamy soils are efficiently lysed in approximately 60 seconds with the FastPrep 96 Instrument from MP Biomedicals The FastPrep 96 Instrumentisa high throughput homogenizer developed to disrupt thoroughly any tissues and cells through the simultaneous bead beating and impaction of specialized Lysing Matrix beads on the sample material The FastPrep 96 Instrument uses a linear vertical bidirectional motion providing an extremely quick and highly reproducible homogenization that surpasses early generation homogenizers as well as traditional extraction methods using enzymatic digestion sonication blending douncing or vortexing Samples are placed into 1 2 ml size tubes of a FastDNA 96 Lysing Matrix Rack 96 deep well plate containing 0 5 mm lysing matrix beads The lysing matrix beads which are specially stabilized Zirconium oxide particles are designed to efficiently
7. DNA from various soil samples extracted with the FastDNA 96 Soil Microbe Kit 10 of the DNA isolated from 135 mg soil samples was loaded on a 0 8 agarose ethidium bromide gel 1 kb Ladder Marker NEB Soil samples include Lane 1 Sand Lane 2 Sandy Clay Lane 3 Sandy Loam Lane 4 Coarse Sandy Loam Lane 5 Fine Gravel Sandy Loam DNA ranges from 4 20 kb FastDNA 96 Soil Mi Figure 2 PCR results of the DNA extractions from Figure 1 respectively The PCR was executed using DNA primers specific to prokaryotic 16S rRNA 7 Recommended Reference Format for Publications DNA was isolated from specific sample using the FastDNA 96 Soil Microbe DNA Kit and the FastPrep 96 Instrument MP Biomedicals LLC Santa Ana CA 8 References Soil and Plant Laboratory Inc PO Box 117744 Santa Ana CA 92711 9 Technical Support For technical support with this product please contact our MP Biomedical s Technical Support Team at 1 800 854 0530 by email at biotech mpbio com or visit us online at www mpbio com for live support For our European customers please contact our European Technical Support Team at 00 800 7777 9999 or by email at techsup eur mpbio com 12 10 Product Use Limitation 8 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 disea
8. nd continue 2 Load the FastDNA 96 Lysing Matrix Rack into the FastPrep 96 Instrument and process the samples A single 60 second run at a speed setting of 1600 rpm is sufficient to lyse almost all samples If additional processing time is required over 5 minutes the FastDNA 96 Lysing Matrix Rack should be incubated on ice for at least 2 minutes between successive runs to prevent overheating the samples 3 Place the FastDNA 96 Lysing Matrix Rack in a microplate centrifuge adaptor and spin at 3 500 5 000 x g for 5 minutes NOTE Extending centrifugation to 10 15 minutes can enhance elimination of excessive debris from soil samples or from cells with complex cell walls 4 Transfer up to 250 ul of supernatant to the wells of a clean Deep Well Plate 5 Add 750 ul of Soil DNA Binding Solution to the supernatant in each well of the Deep Well Plate Cover the wells of the Deep Well Plate completely with the supplied Foil Plate Cover Place the samples on a plate shaker or vortexer and shake mix for 2 minutes FastDNA 96 Soil N 6 Centrifuge the Deep Well Plate for 5 minutes at 3 500 5 000 x g 7 Place the MP 96 Binding Plate on top of a supplied Collection Plate Remove the foil from the Deep Well Plate and transfer 500 ul of each supernatant to the wells of the MP 96 Binding Plate Centrifuge the stacked plates for 5 minutes at 3 500 5 000 x g 8 Discard the flow through from the Collection Plate and re u
9. se Repeat Step 7 until all of the supernatant has been carefully transferred to the binding plate 9 Continue to re use the Collection Plate by placing it beneath the MP 96 Binding Plate To the wells of the MP 96 Binding Plate add 200 ul of the Binding Plate Pre Wash Buffer Centrifuge the stacked plates for 5 minutes at 3 500 5 000 x g 10 To the wells of the MP 96 Binding Plate add 500 ul of Soil DNA Wash Buffer Centrifuge the stacked plates for 5 minutes at 3 500 5 000 x g 11 Stack the MP 96 Binding Plate atop a clean Elution Plate Add 50 100 ul of Elution Buffer directly to the matrix inside the wells of the MP 96 Binding Plate Centrifuge the stacked plates for 5 minutes at 3 500 5 000 x g NOTE DNA isolation is complete at this point if bacterial of fungal broth cultures were sampled Continue with protocol only if sampling from soil or sludge 12 Stack a prepared MP 96 Inhibitor Removal Plate see Section 3 2 for details atop a clean Elution Plate Transfer the eluent from Step 11 to the wells of the MP 96 Inhibitor Removal Plate Centrifuge the stacked plates for 5 minutes at 3 500 5 000 g 10 13 Eluted DNA is now ready for PCR and other downstream applications To store the samples cover the Elution Plate with the supplied Foil Plate Cover Store samples at 4 C until use or at 20 C for extended periods 6 Example Data DNA Isolation from Soil Samples and Gel Electrophoresis Figure 1
10. ses 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 there of 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 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 GeneClean and FastPrep are registered trademarks of MP Biomedicals LLC 13 FastDNA 96 Soil Mi Other Fa
11. stPrep Kits Available FastDNA Kit and FastDNA Spin Kit Cat N 6540 400 Cat N 6540 600 respectively 100 preps Lyse and isolate DNA in less than 30 minutes Plant animal yeast fungal and microbial samples No hazardous organic reagents required SPIN filters streamline silica handling FastDNA Spin Kit FastDNA Spin Kit for Feces Cat N 6570 200 50 preps Lyse and isolate PCR ready DNA in less than 30 minutes Variety of stool sample types No hazardous organic reagents required SPIN filters streamline silica handling FastDNA 50ml Spin Kit for Soil Cat N 6560 600 10 preps Process low microbial containing soil samples Lyse and isolate PCR ready DNA in less than 60 minutes Variety of soil and environmental sample types No hazardous organic reagents required SPIN filters streamline silica handling FastRNA Pro Blue Kit Cat N 6025 050 50 preps For use with gram positive and gram negative bacteria e Lyse up to 1010 cells per 2ml tube Lysis and isolation with single phase organic solution in less than 0 minutes FastRNA Pro Red Kit Cat N 6035 050 50 preps For use with yeast cells and fungal tissue 14 e Lyse up to 1010 cells per 2ml tube Lysis and isolation with single phase organic solution in less than 90 minutes FastRNA Pro Green Kit Cat N 6045 050 50 preps For use with all plant and animal samples e Lyse 50 100 mg tissue per 2ml tube
12. te Prep Solution to the wells by puncturing the foil covering with the pipette tip Incubate at room temperature for 5 minutes then centrifuge the stacked plates at 3 500 x g for 5 minutes The MP 96 Inhibitor Removal Plate is now ready for sample preparation 4 Safety Precautions Some of the supplied kit reagents 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 at all times when using this product Consult the Material Safety Data Sheet at www mpbio com for additional details This product is for research purposes only 5 Protocol IMPORTANT NOTE BEFORE STARTING PROTOCOL When isolating DNA from soil samples the wells of the MP 96 Inhibitor Removal Plate must be prepared for proper use Do not attempt this assay without first preparing the MP 96 Inhibitor Removal Plate or humic substance contamination may occur See Section 3 2 above for instructions on preparing the MP 96 Inhibitor Removal Plate the tubes of a FastDNA 96 Lysing Matrix Rack add up to 1 mg of soil and 400 ul of Lysis Buffer per well Re cap the 135 tubes Optional If broth culture cell lysis is desired add 10 20 mg of wet weight bacterial or fungal cells resuspended in up to 50 XI of sterile water or sterile PBS to the tubes of the FastDNA 96 Lysing Matrix Rack Add Lysis Buffer a
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