96 well Plant Genomic DNA Purification Kit

Transcription

1 96 well Plant Genomic DNA Purification Kit Cat. #: DP / DP Size: 2 x 96 / 8 x 96 Reactions Store at RT For Research Use Only

2 Description : The 96 well Plant Genomic DNA Purification Kit is designed for automated high-throughput and fast isolation of genomic DNA from various kinds of plants and fungi samples, especially for polysaccharide-rich plants and for plant tissue rich in secondary metabolites. The procedure processes using both vacuum-driven transfer and centrifugation, and 8 to 16 μg of genomic DNA can be obtained from 10 mg of tissue for each well. Components of the kit : DP DP Extraction solution A 110 ml 300 ml 2. * Extraction Solution B powder concentrate 2.76 g (Add 12ml sterile water) 11.5g (Add 50ml sterile water) 3. * RNase A powder 200 mg 200 mg x4 ( green cap ) 4. Precipitation 40 ml 120 ml Solution 5. *Binding Solution 75ml ( add 150ml of Ethanol before use) 300 ml ( add 600ml of Ethanol before use ) 6. Wash solution 100 ml ( add 400ml of Ethanol before use ) 200 ml x2 ( add 800 ml of Ethanol before use ) 7. Elution Solution 60 ml 240 ml well Filter Plates 2 pcs 8 pcs well Binding Plates 2 pcs 8 pcs ml Deep Well Plates 4 pcs 16 pcs 11. Adhesive film 12 pcs 48 pcs Upon storage at low temperature, Extraction solution A may form SDS precipitate, which can be easily dissolved by incubation at 35 C. Store the lyophilized RNase A at -20 C and store all other kit components at RT

3 Materials to be supplied by the user: For tissue grinding: mortar and pestle. Liquid nitrogen 100% Ethanol Collection plates 2.2 ml Deep well plate (optional) Before using the kit : To make Extraction Solution B Add sterile water(dp :12 ml, DP :50 ml) into Extraction Solution B powder concentrate, vortex for 1 min and incubate at 50 C in water bath till complete dissolution. Store the solution at -20 C. Add Ethanol (DP : 150ml, DP :300 ml) into Binding Solution and mix well. Store at RT. Add Ethanol (DP : 400ml, DP :800 ml) into Wash Solution and mix well. Store at RT. Dissolve RNase A powder in 1ml sterile water and store the solution at -20 C. Thaw Extraction Solution B before homogenizing tissue. Preheat water bath or heating block to 65 C. General Procedures: Homogenize the plant tissue (< 50 mg) in liquid nitrogen to fine powder using mortar and pestle. Prevent sample from thawing. * Use of more sample than recommended amount may clog the column and reduce the yield and quality. * For optimum yield, grind the tissue thoroughly. DNA in insufficiently ground tissue may be inaccessible to the reagents. *For intact DNA, use fresh tissues or those frozen (immediately after harvest) in liquid nitrogen and subsequently stored at

4 A Centrifuge Protocol: 1. Immediately transfer the ground material to collection plate or 2.2 ml Deep Well Plate(GeneMark, Cat. No.: KG4231), and add 360 μl Extraction Solution A, 40 μl Extraction Solution B and 4 μl RNase A solution. Vortex vigorously for 5~10 sec. During vortex the plate should be covered with Adhesive film. ** Do not premix these solutions.** 2. Incubate at 65 C in water-bath or heating block for 10~20 min. Mix by inverting the plate from time to time. The plate should be covered with Adhesive film during heating. 3. Add 130 μl Precipitation Solution to each well. Mix by inverting the plate, and incubate on ice for 5 min. * The solution will become cloudy because of precipitation of detergent, proteins, polysaccharides and secondary metabolites. 4. Centrifuge at 3000 x g for 5 min at RT. 5. Place a 96 -Well Filter Plate(not 96-well Binging Plate) on the top of a new 1.6 ml Deep Well Plate. Collect the supernatant into the 96 -Well Filter Plate. Centrifuge at 3000 x g for 2 min. * Most tissue clumps and cell debris will be removed by Filter plate, but small amount will pass through and form a pellet in the 1.6 ml Deep Well Plate. Do not disturb the pellet. 6. Add 1.5 volume of Binding Solution (with Ethanol) into 1.6 ml Deep Well Plate and mix thoroughly by inverting. The plate should be covered with Adhesive film during inverting the plate. * For Example: For 500 μl lysate, add 750 μl Binding Solution/Ethanol mixture. * After addition of Binding Solution / Ethanol a stringy precipitate may form. This will not affect the DNA isolation. Load the solution and precipitate into the Binding plate in the next step. 7. Transfer < 650 μl of the mixture including any precipitate into each well of the 96-well Binding Plate with collection plate and centrifuge at 3000 x g for 2 min

5 Discard the flow-through. 8. Repeat step 7 if there is any remaining mixture. 9. Discard the filtrate and add 600 μl of Wash Solution and seal the plate with the same Adhesive film. Centrifuge at 3000 x g for 2 min. Repeat the step two times more. 10. Discard the filtrate and seal the plate with the same Adhesive film.centrifuge the plate at 3000 x g for 2 min to remove any residual traces of ethanol. 11. Transfer the 96-Well Binding Plate on the top of a new 1.6 ml Deep Well Plate. Tear the Adhesive film from the 96-Well Binding Plate and incubate the plate at 45~60 C oven for 5 min to evaporate all of the ethanol. 12. Add 100~200 μl of Elution Solution or H 2 O (ph ) into the center of the wells of the 96-Well Binding Plate and seal the plate with the same Adhesive film. Let stand for 1-2 min. 13. Centrifuge at 3000 x g for 5 min to elute the DNA. 14. Seal the 1.6 ml Deep Well Plate with the new adhesive film and store the plasmid DNA at -20 C. * Repeating elution once will increase DNA yield by 10~15%, though DNA will be diluted. B. Vacuum Protocol 1. Immediately transfer the ground material to collection plate or 2.2 ml Deep Well Plate(GeneMark, Cat. No.: KG4231), and add 360 μl Extraction Solution A, 40 μl Extraction Solution B and 4 μl RNase A solution. Vortex vigorously for 5~10 sec. During vortex the plate should be covered with Adhesive film. ** Do not premix these solutions.** 2. Incubate at 65 C in water-bath or heating block for 10~20 min. Mix by inverting the plate at frequent intervals. During heating, the plate should be covered with Adhesive film. 3. Add 130 μl Precipitation Solution, mix by inverting the plate. Incubate on ice for 5 min

6 *The solution will become cloudy because of precipitation of detergent, proteins, polysaccharides and secondary metabolites. 4. Centrifuge at 3000 x g for 5 min at RT. 5. Place the adapter in the base of the Vacuum manifold (GeneMark, Cat No.: GM-Vac400) and place a 1.6 ml Deep Well Plate on the adapter. Place the 96-Well Filter plate(not 96-well Binding plate) on the top of the vacuum manifold. 6. Collect the supernatant into 96-Well Filter plate. Apply vacuum until all the lysate have passed through. *While most tissue clumps and cell debris will be retained by the Filter plate, small amount may pass through and form a pellet in the Collection plate. If so, do not disturb the pellet. 7. Add 1.5 volume of Binding Solution (with Ethanol) into 1.6 ml Deep Well and mix thoroughly by inverting the plate. The plate should be covered with Adhesive film during inverting. *For example: For 500 μl lysate, add 750 μl Binding Solution/Ethanol mixture. *After addition of Binding Solution/Ethanol may form, which does not affect the DNA purification process. 8. Place a Waste tray in the base of the vacuum manifold and place a 96-Well Binding Plate on the top of the Vacuum manifold. Carefully pipette < 650 μl of the mixture including any precipitate to the 96-Well Binding Plate. Apply vacuum until all the lysate have passed through. Switch off vacuum. 9. Repeat step 8 if there is any remaining mixture. 10. Add 600 μl Wash Solution to each well. Apply vacuum until all the solution have passed through. Switch off the vacuum. Repeat the step two times more. 11. Discard the flow-through. Apply vacuum for another 2 min to remove residual trace of ethanol. 12. Tear the Adhesive film from the 96-Well Binding Plate and incubate the plate at C oven for 5 min to evaporate all of the ethanol. 13. Place the adapter inside the base of the Vacuum Manifold and place a new

7 ml Deep Well Plate on the adapter. Place the 96-Well Binding Plate on the top of the Vacuum Manifold. 14. Add 100~200 μl of Elution Solution or H 2 O (ph ) into the center of the wells of the 96-Well Binding Plate and seal the plate with the Adhesive film. Let stand for 1-2 min. 15. Switch on the vacuum. Apply vacuum for 2 min until DNA elution is complete. 16. Seal the 1.6 ml Deep Well Plate with the new Adhesive film and store the DNA at -20 C. * Repeating elution once will increase DNA yield by 10~15%, though DNA will be diluted

8 Troubleshooting Guide Comments and suggestion Binding plate is clogged a) Too much sample was used. b) Sample was not lysed completely. Poor DNA yield a) Sample may be old or degraded. b) Wash Solution was not prepared properly. c) Sample was not lysed completely d) DNA was not eluted properly Reduce the sample volume 1) Grind the tissue as fine as possible. 2) Do not premix the solutions used in Step 1. 3) Mix frequently during digestion to ensure more efficient lysis or extend the incubation time in the Step 2. Yields will vary among different plant tissues and plant species. Use younger leaves or tissues. If samples are to be stored for future use, flash-freeze in liquid nitrogen and store at -70 C. Make sure to add ethanol to Wash Solution before use 1) Grind the tissue as fine as possible. 2) Do not premix the solutions used in Step1. 3) Mix frequently during digestion to ensure more efficient lysis or extend the incubation time in the Step2. 1) DNA should be eluted only in low-salt Solution [e.g., 10 mm Tris-HCl, ph 8.5 (Elution solution) or water]. Elution efficiency is dependent on ph. The maximum efficiency is achieved between ph

9 7.0 and 8.5. When using water for elution, make sure that the ph is within this range. 2) Ensure that Elution Solution is added at the center of the membrane and is completely absorbed. 3) Allow Elution Solution to incubate in the column for longer time or elute twice to increase the DNA recovery. e) DNA is sheared or degraded 1) Avoid extensive pipetting and vortexing when lysis/homogenization to prevent shearing of DNA. 2) Avoid repeated freezing and thawing of DNA samples

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