FastDNA® Kit - MP Biomedicals
Contents
1. seeds Homogenization in the FastPrep Instrument with Lysing Matrix A takes place in the presence of sample specific Cell Lysis Solutions CLS For plant tissues CLS VF is used in conjunction with a Protein Precipitation Solution PPS Yeast algae and fungi are lysed in the presence of CLS Y For all other samples CLS TC is used during sample lysis For maximum flexibility all buffers are provided in the kit Following lysis samples are centrifuged to pellet debris and lysing matrix DNA is purified from the supernatant with a silica based GENECLEAN procedure Eluted DNA is ready for digestion electrophoresis PCR and any other desired application www mpbio com FastDNA Kit 2 Kit Components and User Supplied Materials 2 1 FastDNA Kit Components Lysing Matrix A 100x 2 0 ml tubes 4 Ceramic Spheres 100 spheres Binding Matrix 66 ml Concentrated SEWS M 12 ml DES 25 ml CLS VF 90 ml PPS 25 ml CLS TC 110 ml CLS Y 110 ml User manual each MSDS each Certificate of Analysis each 2 2 User Supplied Materials FastPrep Instrument see Section 8 Microcentrifuge that can freely spin 2 0 ml tubes Microcentrifuge tubes 2 0 ml and 1 5 ml Rotator or low speed vortex Optional SPIN Filters and Catch Tubes 100 Cat 2080 800 6 www mpbio com 3 Important Considerations Before Use 3 1 Preparation of SEWS M Wash Solution The FastDNA Kit contains a bottle with 2 ml of Concentrated SEWS M Was2. suspension volume of 200 pl NOTE See section 3 3 for other important guidelines www mpbio com FastDNA Kit 2 Add appropriate Cell Lysis Solution CLS according to table below Processing Tissue From Add to Sample Tube Plant tissue 800 ul CLS VF and 200 pl PPS Animal tissue cultured cells insects bacteria bone etc 1 0 ml CLS TC Yeast algae or fungi 1 0 ml CLS Y Homogenize in the FastPrep Instrument for 40 seconds at a speed setting of 6 0 Centrifuge at 14 000 x g for 5 10 minutes to pellet debris Transfersupernatant 700 800 ul toa2 0 ml microcentrifuge tube and add an equal volume of Binding Matrix Invert to mix NOTE It is important to use a tube that is large enough to allow room for complete mixing of the entire volume during the course of the next step Tubes with conical bottoms are not recommended A 2 0 ml microcentrifuge tube works well at this step Incubate with gentle agitation for 5 minutes at room temperature on a rotator NOTE A low speed vortex may be used at this point but care must be taken not to shear the DNA Centrifuge at 14 000 x g for minute to pellet the Binding Matrix Discard the supernatant Add 500 ul prepared SEWS M and gently resuspend the pellet using the force of the liquid from the pipet tip www mpbio com NOTE Ensure that ethanol has been added to the Concentrated SEWS M See section 3 1 9 Centrifuge at 14 000 x g for minute a
3. website at http www qbiogene com fastprep www mpbio com 8 Related Products Description FastPrep 24 Instrument FastPrep FPIO0A Instrument FastPrep FP120A Instrument FastPrep FP220A Instrument FastDNA SPIN Kit FastDNA SPIN Kit for Soil FastRNA Pro Soil Direct Kit FastRNA Pro Soil Indirect Kit FastRNA Pro Red Kit Yeast amp Fungus FastRNA Pro Green Kit Plant amp Animal FastRNA Pro Blue Kit Bacteria FastProtein Blue Matrix FastProtein Red Matrix Lysing Matrix A Lysing Matrix A Lysing Matrix A SPIN Modules Size 100 230V 100V 120V 220V 100 preps 50 preps 50 preps 50 preps 50 preps 50 preps 50 preps 50 preps 50 preps 50 x 2 ml tubes 100 x 2 ml tubes 500 x 2 ml tubes 100 Filters and Catch Tubes www mpbio com Catalog 6002 500 6001 100 6001 120 6001 220 6540 600 6560 200 6070 050 6075 050 6035 050 6045 050 6025 050 6550 400 6550 600 69 10 050 6910 100 6910 500 2080 800 FastDNA Kit 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 item
4. DNA from Dry Sample 3 5 Co Purification of RIN reke 3 6 Use of optional SPIN filtere Safety Precautions cseeeseteeseeeseeeseesseseneeesetseeeeeetes PROCOCOD RAS T Recommended Reference Format for Publication References 25a sce sae cece earan Related Product sssrini esia Product Use Limitation amp Warranty www mpbio com I Introduction to the FastDNA Kit and the FastPrep Instruments The FastDNA Kit quickly and efficiently isolates genomic DNA from a wide variety of sources Designed for use with the FastPrep Instruments from MP Biomedicals plants animal tissues yeast bacteria algae fungi and many other 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 methods using enzymatic digestion sonication blending douncing and vortexing Samples are placed into 2 0 ml tubes containing Lysing Matrix A irregularly shaped garnet particles and a single 1 4 inch ceramic sphere While almost all samples are easily processed with this pre filled combination additional 4 inch ceramic spheres are provided for hard samples such as bone cartilage or
5. Instruction Manual FastDNA Kit Rapid Isolation of Genomic DNA from One Call Plant and Animal Tissue Bacteria Yeast Algae and Fungi Using the FastPrep and FastPrep 24 Instruments One Source Catalog 6540 400 100 Preps Storage A World of Ambient temperature 15 30 C Biotechnology Reagents Revision 6540 400 O6APR www mpbio com MP Biomedicals 29525 Fountain Parkway Solon OH 44139 tel 1 800 854 0530 fax 1 800 334 6999 FastDNA Kit m Aq D ddi Hg i as 6 gt a By MP Biomedicals Our family of companies includes 101 Systems Q FastDNA Kit Rapid Isolation of Genomic DNA from Plant and Animal Tissue Bacteria Yeast Algae and Fungi Using the FastPrep and FastPrep 24 Instruments Catalog 6540 400 100 Preps Storage Ambient temperature 15 30 C Revision 6540 400 06APR DEH FastDNA Kit TABLE OF CONTENTS or oN gt g Introduction to the FastDNA Kit and the FastPrep Tarallu lU DN HH HHHHHHHDD nnn Kit Components and User Supplied Materials 2 1 FastDNA Kit ComponentsS eree 2 2 User Supplied MaterialS E Important Considerations Before Use 3 1 Preparation of SEWS M Wash Solution 3 2 Precipitate Material in CLS TC Buffer 3 3 Sample Lysis with the FastPrep Instrument 3 4 Recovery of
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7. ation is necessary the final eluted DNA can be treated with RNase as per the manufacturer s protocol 8 www mpbio com 3 6 Use of optional SPIN filters MP Biomedicals strongly recommends the use of SPIN filters during the silica based GENECLEAN procedure for final purification of genomic DNA Although the traditional bind wash elute procedure is fine for almost all standard applications the use of SPIN filters eliminates accidental transfer of silica particles and can also lead to increased purity of the final sample Spin filters and catch tubes can be purchased separately Cat 2080 800 or a FastDNA SPIN Kit can be purchased Cat 6540 600 4 Safety Precautions Binding Matrix contains 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 enclosed Material Safety Data Sheet for additional details 5 Protocol Add sample to Lysing Matrix A tube Place up to 100 200 mg tissue fresh frozen dried etc or 200 ul of cells suspended in water or isotonic saline solution For bacteria yeast algae or tissue culture cells grown in suspension Centrifuge a sufficient volume of culture to provide a pellet size of 50 00 mg wet weight or up to l0 bacteria 0 yeast algae or 10 mammalian cells Resuspend pellets in water or isotonic saline to give a maximum
8. h Solution Before using this solution 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 Precipitation in CLS TC Buffer If the Fast DNA Kit was shipped or stored at a low temperature a harmless precipitate may form in the CLS TC Buffer If a precipitate is seen incubate the bottle in a 45 55 C water bath for several minutes and mix to bring the precipitate back into solution Allow solution to cool to room temperature 3 3 Sample Lysis with the FastPrep Instrument The fill volume in the lysing matrix tube after the addition of the Cell Lysis Solution to the sample should allow sufficient air space in the sample tube for efficient FastPrep Instrument processing MP Biomedicals recommends using 100 200 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 Instruments The use of other products with the FastPrep Instruments is not recommended and may result in sample loss or inst
9. nd discard the supernatant NOTE If using a SPIN filter transfer resuspended Binding Matrix to a SPIN Filter Centrifuge at 14 000 x g for minute and discard content of the Catch Tube Centrifuge a second time at 14 000 x g for minute and remove residual liquid with a small pipet tip NOTE If using a SPIN Filter centrifuge a second time at 14 000 x g for minute and replace the Catch Tube with a new clean tube Elute DNA by gently resuspending Binding Matrix in 100 ul of DES Incubate for 5 minutes at 55 C in a heat block or water bath Centrifuge at 14 000 x g for minute Transfer eluted DNA to the clean microcentrifuge tube DNA is now ready for downstream applications Store at 20 C for extended periods or 4 C until use NOTE Avoid transferring particles of Binding Matrix with eluted DNA To avoid shearing transfer the DNA carefully using a pipet tip NOTE If using a SPIN Filter centrifuge at 14 000 x g for minute to bring eluted DNA into the clean catch tube DNA is now ready for downstream applications Store at 20 C for extended periods or 4 C until use www mpbio com FastDNA Kit 6 Recommended Reference Format for Publications DNA was isolated from specific sample using the FastDNA Kit and the FastPrep Instrument MP Biomedicals Irvine CA 7 References A wide variety of references for lysis and purification with FastPrep products can be found on the Qbiogene
10. rument failure www mpbio com FastDNA Kit A single 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 A tube for at least 2 minutes between successive FastPrep Instrument homogenizations to prevent overheating the sample and tube MP Biomedicals recommends that all researchers begin the protocol with the Lysing Matrix A as supplied in the kit garnet matrix and single sphere If lysis is inefficient even after multiple runs of 40 seconds an additional 4 inch ceramic sphere provided can be added on top of the sample Depending on the sample lysis and or yield may or may not improve and shearing of existing genomic DNA may begin to occur Samples with 2 spheres should be processed carefully in order to balance increased yield and lysis against increased DNA shearing by varying speed and or time settings 3 4 Recovery of DNA from Dry Samples To optimize DNA recovery from extremely dry samples leave the lysed sample at room temperature in the Lysing Matrix A tube for an incubation period of 15 minutes to 2 hours after processing in the FastPrep Instrument 3 5 Co Purification of RNA Some tissues i e liver kidney contain very high levels of RNA which may co purify with the genomic DNA If absolute control of RNA contamin
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