FLORACLEAN Plant DNA Isolation Kit

Transcription

1 Instruction Manual FLORACLEAN Plant DNA Isolation Kit Isolate up to 100 µg High Molecular Weight Genomic DNA from a Wide Range of Plant Tissues and Species. DNA is Ready for Restriction Enzyme Digestion, Electrophoresis, or PCR without Organic Extractions One Call One Source A World of Biotechnology Reagents 25 Preps Storage: Ambient (15-30 C) and Refrigerated (2 8ºC) Catalog # Revision # OCT MP Biomedicals Fountain Parkway Solon, OH tel: fax:

2 FLORACLEAN Plant DNA Isolation Kit Lyse BIG and FAST with Interchangeable Adapters! QuickPrep TM Adapter 24 x 2 ml samples TeenPrep TM Adapter 12 x 15ml samples HighPrep TM Adapter 48 x 2ml samples BigPrep TM Adapter 2 x 50 ml samples The FastPrep-24 instrument is delivered with the QuickPrep Adapter 2

3 FLORACLEAN Plant DNA Isolation Kit Isolate up to 100 µg High Molecular Weight Genomic DNA from a Wide Range of Plant Tissues and Species. DNA is Ready for Restriction Enzyme Digestion, Electrophoresis, or PCR without Organic Extractions 25 Preps Storage: Ambient (15-30 C) and Refrigerated (2 8ºC) Catalog # Revision # OCT 3

4 FLORACLEAN Plant DNA Isolation Kit TABLE OF CONTENTS 1. Introduction to the FLORACLEAN Plant DNA Isolation Kit Kit Components and User Supplied Materials FLORACLEAN Kit Components User Supplied Materials Important Considerations Before Use...6 Preparation of Nuclear Buffer Solution Storage Safety Precautions Short Protocol Long Protocol Prepare Tissue Lyse Cellular Membranes Isolate and Wash Nuclei Lyse Nuclei Precipitate DNA Example Data: DNA Isolation from Plant Samples and Gel Electrophoresis Recommended Reference Format for Publication Related Products Product Use Limitation & Warranty

5 1. Introduction to the FLORACLEAN Plant Isolation Kit The FLORACLEAN Plant DNA Isolation Kit is used to efficiently isolate high molecular weight genomic DNA from a wide range of plant tissues and species. Each preparation yields up to 100 µg of genomic DNA from 1.5 gm of starting tissue. Preparation sizes can be scaled up or down. The DNA is isolated from purified nuclei, thus eliminating many cytoplasmic compounds that co-purify with DNA when using other methods of DNA isolation and which can inhibit enzymes. The procedure requires about 2 hours of manipulation and 2-4 hours of incubation time. Resulting DNA is suitable for restriction enzyme digestion or PCR amplification. The procedure does not use organic extractions. The FLORACLEAN Plant DNA Isolation Kit is available with 25-preps. Each kit contains Nuclear Buffer for lysing cellular membranes, chloroplasts, and mitochondria while keeping nuclei intact. Nuclei are lysed with Nuclear Lysis Solution. The kit utilizes Protease Mixx to degrade cellular proteins. This is followed by a proprietary salting-out technique which precludes the need for phenol, chloroform, or other organic extractions. The DNA is precipitated with ethanol, and subsequently pelleted by centrifugation and dissolved in TE. Eluted DNA is ready for digestion, electrophoresis, PCR and any other desired downstream applications. 2. Kit Components and User Supplied Materials 2.1 FLORACLEAN Plant DNA Isolation Kit Nuclear Buffer Concentrate 125 ml CHO powder 107 gm Nuclear Buffer Chaotrope 3.12 ml Nuclear Suspension Solution 45 ml Nuclear Lysis Solution 5 ml Protease Mixx 700 µl Salt-Out Mixture 15 ml Sieve Material 1 each User manual 1 each MSDS (Online: ) 1 each Certificate of Analysis 1 each 5

6 FLORACLEAN Plant DNA Isolation Kit 2.2 User Supplied Materials 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 Sterile water TE (Tris-EDTA Solution) 100% Ethanol 3. Important Considerations Before Use Preparation of Nuclear Buffer Solution The FLORACLEAN Plant DNA Isolation Kit contains buffers that need to be mixed for use in the assay. Dissolve the CHO Powder (107 g) in 400 ml sterile water. Autoclave the solution at 121 C for 15 minutes, and allow solution to cool to room temperature. Next, add the Nuclear Buffer Concentrate (125 ml) to the CHO solution and mix. Add the Nuclear Buffer Chaotrope (3.12 ml) and mix. Finally, add sterile water to a final volume of 625 ml. Label the bottle with the Nuclear Buffer label supplied with the kit. Ensure that the bottle is securely closed to prevent evaporation, and store in the refrigerator (2-8 C). 4. Storage The FLORACLEAN Plant DNA Isolation Kit is shipped at ambient temperature. However, upon arrival store Protease Mixx at 4 C. A few days at ambient temperature does not affect Protease Mixx activity. The Protease Mixx is a suspension. Cloudiness or visible aggregates may appear and can increase with time but do not affect activity. If precipitates are visible in Protease Mixx, remove through centrifugation. 5. Safety Precautions Protease Mixx 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 Material Safety Data Sheet at for additional details. 6

7 6. Short Protocol 1. Grind tissue in liquid nitrogen (optional). 2. Homogenize 1.5 g tissue in 10 ml Nuclear Buffer. 3. Filter homogenate through Sieve Material. 4. Pellet nuclei at 2000 x g for 20 minutes at 4 C. 5. Wash nuclei 3 times with 5 ml Nuclear Buffer. 6. Resuspend washed nuclear pellet in 1.5 ml Nuclear Suspension Solution. 7. Lyse nuclei by addition of 100 µl Nuclear Lysis Solution. 8. Add 25 µl Protease Mixx and incubate at 55 C for 1-4 hours. 9. Add 500 µl Salt-Out» Mixture; ice 15 minutes; pellet and discard precipitate. 10. Add 2 ml water and 8 ml ethanol to precipitate DNA and centrifuge. 11. Dissolve DNA pellets in µl water or TE. 7. Long Protocol 7.1 Prepare Tissue 1. Grind tissue to a fine powder with a mortar and pestle or Waring blender in liquid nitrogen. Allow the liquid nitrogen to evaporate (tissue becomes lighter in color and does not form clumps); however, do not allow the tissue to thaw. Tissue powder can be stored at -70 C indefinitely. 7.2 Lyse Cellular Membranes 2. Add 1.5 g ground, frozen plant tissue (see Note below) to a 40 ml Dounce (or equivalent) homogenizer on ice, add 10 ml cold Nuclear Buffer and homogenize until the tight plunger smoothly passes by the tissue (usually 4-10 passes of plunger past tissue; clearance between tight plunger and wall of homogenizer is enough to allow nuclei to remain intact). The homogenizer can be removed from the ice for a minute or two while this is being done. Note: fresh tissue can also be homogenized without the need to grind as in Step 1. If the tissue is relatively soft (young leaves or flowers, e.g.) the results are equivalent to those where prior grinding is included. 7

8 FLORACLEAN Plant DNA Isolation Kit 3. Fold a 4x4-inch square of Sieve Material (cut from the material supplied) twice to form a 2 inch square four layers thick. Separate one of the layers with gloved fingers, place in a small funnel and pour homogenate through the conical sieve allowing the liquid to pass into a beaker on ice. Do not squeeze the last bit of liquid out of the retained debris. Discard the tissue pieces retained by the sieve. Rinse and blot the fabric for reuse. 7.3 Isolate and Wash Nuclei 4. Pour the strained liquid into centrifuge tubes or bottles and centrifuge at 2,000 x g for 20 minutes at 4 C to pellet nuclei. Discard the supernatant fractions which should be green due to lysis of chloroplasts. 5. Resuspend the nuclei in 5 ml cold Nuclear Buffer either by hand or by gentle vortexing. Pellet nuclei as in Step 4. Repeat Step 5 twice. The nuclear pellet should be grayishwhite and not very viscous. Chloroplast or starch contamination causes a green or viscous nuclear pellet. Cells from some tissues will not lyse as easily as others and the pellet will have a greenish tint but low viscosity. DNA isolated from these pellets appears to be of good quality and will readily cut with restriction enzymes. 7.4 Lyse Nuclei 6. Resuspend the washed nuclear pellet in 1.5 ml Nuclear Suspension Solution and transfer to a 2 ml microcentrifuge tube. 7. Add 100 µl Nuclear Lysis Solution and incubate at 55 C for 30 minutes. 8. Add 25 µl Protease Mixx and continue incubation a 55 C for 1-4 hours. Note: The longer incubation time increases the probability of complete digestion of protein, including self-digestion of protease. 9. Add 500 µl Salt-Out Mixture (split into two microcentrifuge tubes if necessary) and incubate on ice for 15 minutes (or overnight in refrigerator). Centrifuge at high speed in microcentrifuge for 5 minutes to pellet precipitated debris. 8

9 7.5 Precipitate DNA 10. Transfer supernatant (pool if in two tubes) to a sterile 15 ml conical tube with screw cap. Add 2 ml water, mix, and add 8 ml 100% ethanol. Mix by inverting. a. If stringy white DNA precipitate becomes visible, centrifuge for 20 minutes at 2000 x g in a bench top clinical centrifuge with a swinging bucket rotor to pellet precipitated DNA. Discard supernatant, blot walls of tube, and air dry pellet for 15 minutes. (To assist in dissolving DNA do not dry pellet under vacuum). b. If stringy white precipitate is not evident place tube at -70 C for 20 minutes (or -20 C overnight) and centrifuge at 10,000 x g (preferably in a swinging bucket rotor) for 20 minutes. Discard supernatant and air dry pellet as in Step (a), above. 11. Dissolve DNA pellets in l of sterile water or TE at room temperature or overnight at 4 C. DNA isolated from most plant species and tissues by the above procedure will digest easily with restriction enzymes and/or amplify. If, however, a DNA sample is resistant to enzymes, an additional cleaning step with the GENECLEAN SPIN Kit will invariably reverse the inhibition. 8. Example Data: DNA Isolation from Plant Samples and Gel Electrophoresis 9

10 FLORACLEAN Plant DNA Isolation Kit 9. Recommended Reference Format for Publications DNA was isolated from (specific sample) using the FLORACLEAN Plant DNA Isolation Kit (MP Biomedicals, Santa Ana, CA). 10. Related Products Description Size Catalog # FastPrep 24 Instrument V FastPrep Instrument 100V FastPrep FP120A Instrument 120V FastPrep FP220A Instrument 220V FastDNA Kit 100 preps FastDNA SPIN Kit 100 preps FastDNA SPIN Kit for Soil 50 preps FastDNA 50ml SPIN Kit for Soil 10 preps FastRNA Pro Soil-Direct Kit 50 preps FastRNA Pro Soil-Indirect Kit 50 preps FastRNA Pro Red Kit (Yeast & Fungus) 50 preps FastRNA Pro Green Kit (Plant & Animal) 50 preps FastRNA Pro Blue Kit (Bacteria) 50 preps FastProtein Blue Matrix 50 preps FastProtein Red Matrix 50 preps Lysing Matrix E 50 x 2 ml tubes Lysing Matrix E 100 x 2 ml tubes Lysing Matrix E 500 x 2 ml tubes

11 11. Product Use Limitation & 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 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). FastDNA, FastRNA, FastPrep, QBiogene, and BIO 101 Systems are registered trademarks of MP Biomedicals, LLC. 11

12 FLORACLEAN Plant DNA Isolation Kit Take Advantage of FastPrep Kits Ready-to-use Protocols For DNA, RNA And Protein Isolation From Any Sample Rapid and reproducible sample lysis and purification process No cross contamination with the closed lysing matrix tubes Increased yields of high quality DNA, RNA and proteins Integrity and size of DNA, RNA and proteins are retained Nucleic acids and proteins are ready-to-use in downstream application DNA FastPrep Kits PROTEIN RNA FastDNA Kit and FastDNA Spin Kit Cat N Cat N 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 Soil Cat N (100 preps) Lyse and isolate DNA in less than 30 minutes Variety of soil and environmental sample types No hazardous organic reagents required SPIN filters streamline silica handling 12

13 FastRNA Pro Blue Kitt Cat N (50 preps) For use with gram positive and gram negative bacteria Lyse up to cells per 2ml tube Lysis and isolation with single-phase organic solution in less than 90 minutes FastRNA Pro Red Kit Cat N (50 preps) For use with yeast cells and fungal tissue Lyse up to cells per 2ml tube Lysis and isolation with single-phase organic solution in less than 90 minutes FastRNA Pro Green Kit Cat N (50 preps) For use with all plant and animal samples Lyse mg tissue per 2ml tube 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 Cat N respectively (50 preps) Isolate RNA from soil samples (direct kit) and washed soil (indirect kit) in less than 2 hours Variety of soil and environmental sample types RNA protected during and after processing Humic acids reduced to allow uninhibited RT-PCR Includes additional reagents for even further purification if necessary SPIN filters streamline silica handling FastProtein Blue Matrix Cat N (50 preps) - Cat N (100 preps) Release of proteins from gram positive and gram negative bacteria in 40 seconds Protein extracts are ready for immediate electrophoresis or purification Ideal for optimizing induction conditions FastProtein Red Matrix Cat N (50 preps) - Cat N (100 preps) Release of proteins from yeast cells and fungi in 40 seconds Protein extracts are ready for immediate electrophoresis or purification Ideal for optimizing induction conditions 13

14 FLORACLEAN Plant DNA Isolation Kit MP Biomedicals Our family of companies includes: 14

15 Lyse BIG and FAST with Interchangeable Adapters! QuickPrep TM Adapter 24 x 2 ml samples TeenPrep TM Adapter 12 x 15ml samples HighPrep TM Adapter 48 x 2ml samples BigPrep TM Adapter 2 x 50 ml samples The FastPrep-24 instrument is delivered with the QuickPrep Adapter 15

16 Instruction Manual FLORACLEAN Plant DNA Isolation Kit Revision # OCT Worldwide Ordering and Technical Support United States of America Worldwide Headquarters Tel: Toll Free Tel: Fax: Toll Free Fax: Europe Toll Free Phone: Toll Free Fax: Australia MP Biomedicals Australasia Pty Ltd Tel: Fax: Belgium MP Biomedicals Tel: Fax: France MP Biomedicals France Tel: Fax: Germany MP Biomedicals Phone: Fax: Japan MP Bio Japan K.K. Tel: Toll Free Tel: Fax: The Netherlands MP Biomedicals Netherlands Tel: Fax: Serbia MP Global d.o.o. Tel: Fax: Singapore MP Biomedicals Singapore Tel: Fax: Switzerland MP Biomedicals Switzerland Tel: Fax: United Kingdom MP Biomedicals UK Tel: Fax: Canada MP Biomedicals Canada Tel: Fax: Poland MP Biomedicals Poland Tel: Fax: MP Biomedicals Fountain Parkway Solon, OH tel: fax: