FastDNA-96™ Plant & Seed DNA Kit
Contents
1. 2 2 Storage All FastDNA 96 Plant amp Seed 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 Optimization of the Plant Seed DNA Binding Solution Prior to beginning the Kit Protocol in Section 5 the Plant Seed DNA Binding Solution can be optimized for increased reagent performance and yield Add 0 590 v v beta mercaptoethanol 750 ul to the 150 ml bottle of the Plant Seed DNA Binding Solution FastDNA 96 Plan 3 2 Binding Plate Pre Wash Buffer The FastDNA 96 Plant amp Seed 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 3 3 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 sup2. MP Global d o o Tel 381 11 2622 945 Fax 381 11 2623 373 Singapore a MP Biomedicals Asia Pacific Pte Ltd Tel 65 6775 0008 Fax 65 6775 4536 United Kingdom MP Biomedicals UK Tel 0800 282 4 Fax 0800 614 735 www mpbio com MP Biomedicals 29525 Fountain Parkway Solon OH 44139 tel 1 800 854 0530 fax 1 800 334 6999
3. 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 9 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 14 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 conseguence of its use or misuse by the Buyer its employees or others including but not limi
4. 4 y Application Manual Instruction Manual Rapid High Throughput Isolation of PCR Ready Genomic DNA from Plant and Seed Samples using the FastPrep 96 System Catalog 9696 600 2 x 96 Preps Storage Ambient temperature 15 30 C Revision 9696 600 11FEB MP Biomedicals 29525 Fountain Parkway Solon OH 44139 e tel 1 800 854 0530 fax 1 800 334 6999 FastDNA 96 Plant amp Seed DNA Kit FastDNA 96 Plant amp Seed DNA Kit Rapid High Throughput Isolation of PCR Ready Genomic DNA from Plant and Seed Samples using the FastPrep 96 System Catalog 9696 600 2 x 96 Preps Storage Ambient temperature 15 30 C Revision 9696 600 11FEB FastDNA 96 Plant amp TABLE OF CONTENTS 1 Introduction to the FastDNA 96 Plant amp Seed DNA Kit and the FastPrep 96 Instrument 5 2 Kit Components Storage amp User Supplied Materials 6 2 1 FastDNA 96 Plant amp Seed DNA Kit Components 6 2 2 SOli GC cess edd i GU FU GT AYN D 7 2 3 User Supplied Materials 0 0 7 3 Important Considerations Before Use 7 3 1 Optimization of Plant Seed DNA Binding Buffer 7 3 2 Binding Plate Pre Wash Buffer ne 8 3 3 MP 96 Inhibitor Removal Plate Preparation 8 8 0060 9 6 Example Data DNA Isolation from Plant 8 Seed Samples and Gel Electrophoresis ss 11 7 Recommended Reference Format for
5. Publication 14 8 Technical 14 9 Product Use Limitation amp Warranty 14 1 Introduction to the FastDNA 96 Plant amp Seed DNA Kit and the FastPrep 96 Instrument The FastDNA 96 Plant amp Seed DNA Kit quickly and efficiently isolates inhibitor free PCR ready genomic DNA from vegetative samples in approximately 50 minutes Tough to lyse plant samples including stems roots leaves buds flowers fruits and seeds are efficiently lysed in approximately 60 seconds with the FastPrep 96 Instrument from MP Biomedicals The FastPrep 96 Instrument is a 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 guickand 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 2 0 mm lysing matrix beads The lysing matrix beads which are specially stabilized Zirconium oxide particles are designed to efficiently lyse a wide variety of Plantae kingdom specimens while in the presence of a specially formulated lysis solution The F
6. astDNA 96 Plant amp Seed DNA Kit isolates plant source DNA from up to 80 mg samples through a purification process that eliminates PCR inhibitors polyphenols tannins and polysaccharides Organic denaturants or proteinases are not needed with this procedure Purified inhibitor free DNA is eluted in an EDTA free DNA elution solution and is ready for FastDNA 96 M Plant downstream applications including digestion electrophoresis arrays 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 Plant amp Seed 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 Plant amp Seed DNA Kit Components FastDNA 96 Lysing Matrix Rack 2 0 mm Beads 2 x 96 well rack Lysis Buffer 2x 40 ml Plant Seed DNA Binding Solution 150 ml Binding Plate Pre Wash Buffer 50 ml Plant Seed DNA Wash Buffer 100 ml Elution Solution 2x10ml Inhibitor 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
7. e MP 96 Binding Plate add 200 Kl 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 Plant Seed 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 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 10 the stacked plates for 5 minutes at 3 500 5 000 g 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 Plant amp Seed Samples and Gel Electrophoresis Figure 1a Figure 1a samples include Lane 1 A thaliana 2 Juniper Lane 3 Milkweed Leaf Lane 4 Milkweed Leaflet Lane 5 Milkweed Pre Flowering Bud DNA purified from plant a and seed b using the FastDNA 96 Plant amp Seed DNA Kit DNA was isolated from eguivalent amounts of vegetative materials and loaded on a 0 8 agarose ethidium bromid
8. e gel 1 kb Ladder Marker Zymo Research Figure 1b Figure 1b samples include Lane 1 2 Corn Kernel Lane 3 4 Sunflower Seed w o MP 96 Inhibitor w MP 96 Inhibitor Removal Plate Removal Plate Figure 2 12 PCR results of DNA extractions from Arabidopsis thaliana illustrating the effectiveness of PCR inhibitor removal by the MP 96 Inhibitor Removal Plate The PCR was executed using DNA primers specific to a 700 bp amplicon of Chromosome 1 Lanes 1 4 and 5 8 exhibit decreasing volumetric dilutions of the PCR product 0 0 001 respectively Amplicons were loaded on a 0 8 agarose ethidium bromide gel 100 bp Ladder Marker Zymo Research Figure 3 PCR results of DNA extractions from corn kernels The PCR was executed using DNA primers specific to a 450 bp amplicon of mitochondrial DNA Lanes 1 4 exhibit decreasing volumetric dilutions of the PCR product 0 0 001 Amplicons were loaded on a 0 8 agarose ethidium bromide gel 100 bp Ladder Marker Zymo Research FastDNA 96 Pla nt amp 7 Recommended Reference Format for Publications DNA was isolated from specific sample using the FastDNA 96 Plant amp Seed DNA Kit and the FastPrep 96 Instrument MP Biomedicals Santa Ana CA 8 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 biotechempbio com or visit us online at www mpbio com for
9. ficient 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 up to 10 minutes can enhance elimination of excessive debris from fecal samples 4 Transfer up to 250 ul of supernatant to the wells of a clean Deep Well Plate 5 Add 750 ul of Plant Seed DNA Binding Solution to the 9 FastDNA 96 Plant 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 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 use 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 th
10. plied Elution Plate Add 150 ul of Inhibitor Plate 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 plant or seed 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 PCR inhibitor contamination may occur See Section 3 2 above for instructions on preparing the MP 96 Inhibitor Removal Plate 1 To the tubes of a FastDNA 96 Lysing Matrix Rack add up to 80 mg of finely cut plant or seed sample and 400 ul of Lysis Buffer per well Re cap the tubes 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 suf
11. ted 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 15 7 Application Manual Revision 9696 400 11FEB Worldwide Ordering and Technical Support 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 1945 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 Russia MP Biomedicals Russia Tel 7 095 995 2844 Fax 7 095 995 2846 gt Serbia and Montenegro
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