96-Well Plant DNA isolation Mini Kit. perts. Quality Kits made by. User Guide. Cat No: XG

Transcription

1 User Guide Cat No: XG

2 Table of Contents Introduction...2 Storage and stability... 2 Safety information... 2 Materials to be provided by users... 3 Kit contents Trouble shooting guide

3 Introduction XcelGen 96-Well Plant DNA Kit allows rapid and reliable isolation of high-quality genomic DNA in a high-throughput 96-well format from plant tissue samples, with unusually high levels of phenolic compounds and polysaccharide. Up to 80 mg of wet tissue (or 25 mg dry tissue) can be processed in each well in less than 1 hour. The system combines the reversible nucleic acid-binding properties of the bind matrix with the speed and versatility of the 96-Well DNA plate to eliminate polysaccharide, phenolic compounds and enzyme inhibitors from tissue lysates. Purified DNA is suitable for PCR, restriction digestion and hybridization techniques. There are no organic extractions, thus reducing plastic waste and hands-on time to allow up to 96 samples to be processed at one time. The DNA binding capacity per well is 30 g. Materials supplied by user Laboratory centrifuge capable of at least 5,000 x g equipped with swinging bucket rotor. Rotor adapter for deep well microplates Absolute (96%-100%) ethanol Multichannel pipette with tips Incubator or vacuum oven preset at 65 C. -mercaptoethanol. Storage and Stability All components of the 96-Well DNA Kit are stable for at least 12 months from date of purchase when stored at C. Buffer CFP may form precipitates in cool ambient conditions, Warm up the bottle at 37 C to dissolve before use. Store RNaseA at -20 C. Safety Information Buffer CBL and CFP contains chaotropic salts, which may form reactive compounds when combines with bleach, Do not add bleach or acidic solutions directly to the preparation waste, ware gloves and protective eyewear when handling. 02

4 Kit Contents Catalog 96-Well DNA Plate 96- Well 2.2 ml Plate 96- Well 1.6 ml Plate Sealing Film 96-Well Collection Plate Buffer CBL Buffer CFP DNA Wash Buffer* Elution Buffer Instruction Booklet XG ml 260 ml ml 100 ml 1 XG x 440 ml 3 x 450 ml 10 x 100mL 450 ml 1 * DNA Wash Buffer Add 400 ml (XG ), 400 ml (XG ) (96%-100%) ethanol to each bottle before use. 03

5 Protocol for Extracting Total RNA From Plant Tissue Grind sample with pestles A. Dry Specimens Drying allows storage of field specimens for prolonged periods of time prior to processing. Samples can be dried with enough allochroic silicagel and stored at room temperature. To prepare dried samples, place ~25 mg of dried tissues into a microfuge tube (1.5 ml tubes are recommended) and grind using a pellet pestle. Disposable Kontes pestles work well. For critical work such as PCR and cloning, pestles are best used a single time then soaked in a dilute bleach solution immediately after use until clean. Disposable pestles may be autoclaved several times. A fine powder will ensure optimal DNA extraction and yield. B. Fresh/Frozen Specimens Due to the tremendous variation in water and polysaccharide content of plants, sample size should be limited to 80 mg from beginning. It is very important not overload the 96- Well plate. Too much starting material will decrease the yield and purity due to the inefficient lysis. We recommend starting with 80 mg tissue at first. If results obtained are satisfactory, then increase amount of starting material to optimal level. Best results are obtained with young leaves or needles. Various means of sample disruption can be used for this kit, such as beads, pestles, but use of liquid nitrogen is recommended with each. For example: To prepare samples collect tissue in a 1.5 ml or 2 ml microfuge tube and freeze by dipping in liquid nitrogen with a pair of tweezers to fill the tube. Grind the tissue using disposable Kontes pellet pestles. For critical work such as PCR and cloning, pestles oare best used a single time then soaked in a dilute bleach solution immediately after use until clean. Disposable pestles may be autoclaved several times. For standard Southern analysis, the same pestle can be reused several times to grind multiple tissue samples by rinsing with ethanol and carefully wiping the surfaces clean between samples. Transfer the ground sample into a 96-well tube rack or deep well plate. Note: Do not allow the sample to thaw during handling and weighing. To prevent sample from thawing, keep the rack or plate on a bed of dry ice. 04

6 Disrupting Samples With Beads Mill Fresh or dried leaves samples can be effectively disrupted and homogenized by rapid agitation in the presence of beads. Leaf samples and beads are added to each well of a 96-well tube rack or deep well plate. The racks or plates are fixed into clamps on a mixer mill. Leaf samples are disrupted and simultaneously homogenized with the shearing and crushing action of the beads. 1. After disrupting sample material, measure up to 25 mg dry powdered leaves or 80mg fresh (or frozen) leaves and place into 96-Well 1.6 ml Plate(supplied). Add 1 ml Buffer CBL/20 L -mercaptoethanol.to each sample. Seal the wells with Sealing Film for 96-Well 1.6 ml Plate (supplied). Vortex briefly and shaking the plate (side to side) for 10 times. 2. Incubate at room temperature for 2 minutes. Note: Incubate no more than 5 min. 3. Centrifuge 96-Well 1.6 ml Plate for 5 minutes at 4,000 x g. 4. Remove the Sealing Film,and discard the supernatant carefully and completely. 5. Add 600 L Buffer CFP/10 L -mercaptoethanol and 8 L RNase A to each sample. Seal the wells with the new Sealing Film. 6. Vortex sharply for 1 min and incubate at room temperature for 2 minutes. 7. Centrifuge 96-Well 1.6 ml Plate for 10 minutes at 4,000 x g. Optional: 96-well Lysate Clearance Plates are available for use with this kit at this step. Optionally, desired volume of solution in Step 5 can be transferred to the 96 Filter Plate to be placed over a new 96-Well 1.6 ml Plate and centrifuged at 3,000-5,000 xg for 5 min. Following centrifugation the protocol would continue at Step Remove and discard the sealing Film, carefully transfer 500 L of each supernatant to a new 96-Well 1.6 ml Plate. If less than 500 L of supernatant is recovered, adjust volume of ethanol in Step Add 0.5 volume(250 L for 500 L) of ethanol. Seal the wells with the new Sealing Film. 10. Vortex or shake 96-Well 1.6 ml Plate side to side somewhat vigorously for 20 05

7 seconds. Centrifuge the 96-Well 1.6 ml Plate briefly to collect any liquid from the Sealing Film. Allow the centrifuge to reach 3000 rpm, and stop the centrifuge. Do not prolong this step. 11. Place 96-Well DNA Plate on top of 96-Well 2.2 ml Plate. Carefully transfer cleared lysate into each well of 96-Well DNA Plate. Be careful not to spill sample liquid onto the rims of the wells during the transfer. Seal the wells with the new Sealing Film. 12. Centrifuge at 3,000-5,000 x g for 5 minutes or until all the sample passes through the 96-Well DNA Plate. 13. Remove the Sealing Film and discard the flow-through from 96-Well 2.2 mlplate. Reassemble the 96-Well DNA Plate with 96-Well 2.2 ml Plate. 14. Add 800 L DNA Wash Buffer to each well of the 96-Well DNA Plate. Seal the plate with new Sealing Film. Centrifuge at 3,000-5,000 x g for 5 minutes. 15. Repeat Step 14 once. 16. Centrifuge 96-Well DNA Plate without Sealing Film at 5,000 x g for 25 minutes to dry the plate. Drying the plate completely is critical for removal of residual ethanol that will otherwise interfere with downstream applications. 17. To elute the DNA, place the 96-Well DNA Plate in right orientation on the 96-Well Collection Plate,add 100 L Elution Buffer or TE(pre-heated at 65 C) to each sample. Seal the plate with a new Sealing Film, Incubate for 8 minutes at 65 C in a incubator. 18. Centrifuge at 4,000 x g for 5 minutes. 19. Seal the 96-Well Collection Plate with a new Sealing Film and store at -20 C. 06

8 Vacuum Manifold Protocol (Optional) 1. Grind sample and prepare the cleared lysate by following Steps Assemble the vacuum manifold : 1) Place the 96-Well DNA Plate on the top part of vacuum manifold. 2). Place the waste collection tray inside base part of the manifold. Seal the unused wells of the 96-Well DNA Plate plate with Sealing Film. 3. Carefully transfer cleared lysate into each well of 96-Well DNA Plate. Be careful not to spill sample liquid onto the rims of the wells during the transfer. Seal the wells with the new Sealing Film. 4. Turn on the vacuum manifold and filter through the sample mixture by vacuum. Turn off the vacuum and load the remainder of the sample into the DNA Plate (if any). Apply the vacuum to draw the sample through the DNA Plate. 5. Add 800 L DNA Wash Buffer into each well of the 96-Well DNA Plate by using a multichannel pipette. Place the plate into vacuum manifold and wash the plate by turning on vacuum. Repeat Step 5 Once. 6. Wash the plate with 400 L absolute (96%-100%) ethanol. Continue the vacuum until the 96-Well DNA plate is completely dried. 7. Remove the 96-Well DNA Plate from manifold and tap hard on a stack of paper towels to remove any ethanol residue. Discard the flow through and collection plate. 8. Note: It is very important to completely dry the 96-Well DNA Plate before elution. If a swing bucket centrifuge with a 96-well plate adaptor is available, centrifuge at 5000 x g for 5 minutes to dry the plate. Or if a vacuum oven is available, place plate in oven set to 70 C for 10 minutes. 9. Place the 96-Well DNA Plate onto a 96-Well Collection Plate. 10. To elute the DNA, add 100 L of preheated Elution Buffer or TE to each well of 96-Well DNA Plate. Incubate for 8 min at 65 C. 11. Assemble the manifold by placing a 96-Well Collection Plate inside the vacuum manifold. Place the 96-Well DNA Plate onto vacuum manifold. Apply the vacuum to elute the DNA into collection plate. 12. Seal the 96-Well Collection Plate with a new Sealing Film and store at -20 C. 07

9 Troubleshooting Guide Problems Possible Reasons Suggestion Clogged column Incomplete lysis Sample too large Mix well after adding Buffer CFP and vortex sharply. It may be necessary to extend incubation time for another 5 min. Scale up the volumes of CFP and ethanol if using more than suggested amouts. Low DNA yield Poor elution Repeat elution or increase elution volume. Incubation of plate at 65 C for 5 min with Elution Buffer may increase yields. Make sure the ph of the water is more than 7.5 Incubate too long in step 2 Incubate 2 min in step 2 Low A260 /A280 ratio Improper washing Extended centrifugation during elution step. Improper mixing with CFP Incomplete cell lysis due to insufficient incubation. Samples are rich in proteins. Silica fine interference DNA Wash Buffer Concentrate must be diluted with absolute (100%) ethanol as specified on Page 3. Resin from the plate may be present in eluate. Avoid centrifugation at speeds higher than specified. Make sure to vortex the sample with Buffer CFP immediately and completely Increase incubation time with Buffer CFP and protease. After applying to wells, wash with 300 L of a 1:1 mixture of Buffer CFP and ethanol and then with DNA Wash Buffer. Remove the silica fines by centrifugation and check the OD again Limited Use and Warranty This product is intended for in vitro research use only. Not for use in human. This product is warranted to perform as described in its labeling and in XcelGens literature when used in accordance with instructions. No other warranties of any kind, express or implied, including, without limitation, implied warranties of merchantability or fitness for a particular purpose, are provided by XcelGen. XcelGens sole obligation and purchaser s exclusive remedy for breach of this warranty shall be, at the option of XcelGen, to replace the products, XcelGen shall have no liability for any direct, indirect, consequential, or incidental damage arising out of the use, the results of use, or the inability to use it product. For technology support or learn more product information, please visit our website at 08

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