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YGPI YGPM Genomic DNA Extraction Kit Midi Maxi Plant ai

VIEWS: 10 PAGES: 1

									                                                                                                                                                                                                                                                                        *
                                                                                                                                                                                                                                                               Store at room
                                                                                                                                                                                                                                                               temperature
                                                                                                                                                                                                                                                                15⁰C~25⁰C




                                                                                                                                                 Genomic DNA Extraction Kit
                                                                                                                                                 [Midi/Maxi] (Plant)
                                                                                                                                                 Protocol Book
                                                                                                                                                 Duo Buffer System
                                            Real Biotech Corp. is a leading R&D based life science solutions provider.
                                            For more information on our extensive and innovative life science range please visit our web site.   Cat.No. YGPI10 / YGPI25 / YGPM10 / YGPM25




                                                                                                                                                                                                          Genomic DNA Extraction Kit [Midi] (Plant)

       Genomic DNA Extraction Kit [Midi](Plant)                                                                                     *
                                                                                                                           Store at room
                                                                                                                           temperature
                                                                                                                            15⁰C~25⁰C
                                                                                                                                                 Description
                                                                                                                                                 Plant Genomic DNA Midi Kits provide a fast and simple method to isolate total DNA (genomic DNA,
                                                                                                                                                 mitochondrial and chloroplast) from plant tissue and cells. Samples are homogenized under liquid nitrogen
                                                                                                                                                 followed by lysis buffer incubation. The lysate is treated with RNase A to degrade RNA and spun through a filter
                                                                                                                                                 column to remove cell debris and salt in the solution. In the presence of binding buffer and chaotropic salt, the
       Cat.No. YGPI10                               Cat.No. YGPI25                                                                               genomic DNA in the lysate binds to the glass fiber matrix in the spin column. The contaminants are washed with
                                                                                                                                                 an ethanol based wash buffer and the purified genomic DNA is eluted by low salt elution buffer or water. The
       10 midi preps / kit                          25 midi preps / kit                                                                          purified genomic DNA is ready for PCR, real-time PCR, Southern blotting and RFLP.
       GP1 Buffer: 25 ml                             GP1 Buffer: 65 ml
       GPX1 Buffer: 25 ml                            GPX1 Buffer: 65 ml                                                                            Protocol Modifications (Duo 2 Buffer System)
                                                                                                                                                 Plant species are extremely diverse in their metabolic components. Large amounts of polysaccharides,
       GP2 Buffer: 6 ml                              GP2 Buffer: 15 ml                                                                             carbohydrates, lipids, polyphenols and proteins may be distributed throughout the plant tissue. These
       GP3 Buffer(concentrated): 15 ml *             GP3 Buffer(concentrated): 40 ml *                                                             compounds often interfere with DNA binding and extraction. This characteristic of plants means we offer two
       Wash Buffer (concentrated): 25 ml **          Wash Buffer (concentrated): 100 ml **                                                         lysis buffers for optimum performance according to plant sample type.
       Elution Buffer: 30 ml                         Elution Buffer: 30 ml
       RNase A(10 mg/ml): 275 μl                    RNase A(10 mg/ml): 650 μl
                                                                                                                                                 GP1: The standard protocol utilizes GP1 buffer for lysis of plant samples. This buffer system is suitable for most
                                                                                                                                                 common plant species, ensuring high quality and high yields of DNA.
       Filter Column: 10 pcs                        Filter Column: 25 pcs
       GD-Midi Column: 10 pcs                       GD-Midi Column: 25 pcs                                                                       GPX1: An alternative buffer, GPX1, is also included with this kit. The detergent present in this buffer is more
                                                                                                                                                 effective in dispersing plant samples with large amounts of polysaccharide. For the majority of plant species both
       Sample: 0.5 g of plant tissue                                                                                                             buffers will give similar results. The researcher may try one buffer system first or both in parallel.
       Yield: 30-150 μg                                                                                                                          Quality Control
       Operation time: < 80 min.                                                                                                                 The quality of the Plant Genomic DNA kits are tested on a lot-to-lot basis. The kits are tested by isolation of
                                                                                                                                                 genomic DNA from 250 mg of young leaves.
                                                                                                                                                 Caution
     * Add two times volume of Isopropanol to GP3 buffer : 30 ml/ 80 ml                                                                           The above components contain irritant agents. During the extraction process always wear a lab coat, disposable
    ** Add four times volume of Ethanol to Wash Buffer :100 ml / 400 ml                                                                           gloves and protective goggles.


1                                                                                                                                                                                                                                                                                    2

                                                                                                                                                                                                          Genomic DNA Extraction Kit [Midi] (Plant)
      Midi Protocol                                                                                                                              Wash
                                                                                                                                                 15. Add 5 ml of Wash Buffer (ethanol already added) into the column.
      Tissue Dissociation                                                                                                                        16. Centrifuge at 4,000 X g for 3 minutes.
      1. Cut off 0.25g (up to 0.5g) of fresh or frozen plant tissue or 25mg (up to 50mg) of dried sample.                                         17. Discard the flow-through and place the GD-Midi Column back in the Collection Tube.
      2. Grind the sample under liquid nitrogen to a fine powder. Transfer it into a 15ml centrifuge tube (not provided).                         18. Repeat the Wash step again.
         For some plant samples it is possible to homogenize the sample without the use of liquid nitrogen.                                      19. Discard the flow-through and place the GD-Midi Column back in the Collection Tube.
                                                                                                                                                 20.Centrifuge at 4,000 X g for 10 minutes to dry the column matrix.
      Lysis
      3. Add 2 ml GP1 Buffer (or GPX1 Buffer) and 25 μl RNase A (10 mg/ ml) into the sample tube and mix by vortexing.
          (Do not mix GP1 buffer and RNase A before use.)                                                                                         DNA Elution
      4. Incubate at 65⁰C for 20 minutes. During incubation, invert the tube every 5 minutes. At the same time, preheat required Elution         Standard elution volume is 0.5 ml. If only small samples are available, reduce the elution volume (150-300 μl) to increase DNA
         Buffer (1 ml per sample) at 65⁰C.                                                                                                        concentration. If higher DNA yield is required, repeat the DNA Elution step to increase DNA recovery and bring the total elution
      5. Add 0.5 ml GP2 Buffer and mix by vortexing.                                                                                              volume to 1 ml.
      6. Incubate at ice for 5 minutes.
      7. Place a Filter Column in a 50 ml centrifuge tube. (not provided)                                                                        21. Transfer dried GD-Midi Column into a clean 50 ml centrifuge tube (not provided).
      8. Apply the mixture from previous step to the Filter Column. Centrifuge at 4,000 X g for 5 minutes.                                       22. Add 0.5 ml of preheated Elution Buffer into the center of the column matrix.
      9. Discard the Filter Column and carefully transfer clarified supernatant in collection tube to a new 15 ml centrifuge tube                 23. Stand for 5 minutes until Elution Buffer absorbed by the matrix.
         (not provided).                                                                                                                         24. Centrifuge at 4,000 x g for 3 minutes to elute purified DNA.

      DNA Binding
      10. Add 1.5 volumes of GP3 Buffer (isopropanol already added) to the cleared lysate and mix immediately by vortexing for 10
         seconds. For example, add 3.75 ml GP3 Buffer to 2.5 ml of lysate.
      11. Place a GD-Midi Column in a 50 ml centrifuge tube.
      12. Apply the mixture (including any precipitate) from previous step to the GD-Midi Column.
      13. Centrifuge at 4,000 X g for 5 minutes.
      14. Discard the flow-through and place the GD-Midi Column back in the collection tube.

3                                                                                                                                                                                                                                                                                    4

                                                                                                                                                                                                           Genomic DNA Extraction Kit [Maxi](Plant)

       Genomic DNA Extraction Kit [Maxi] (Plant)                                                                                    *
                                                                                                                           Store at room
                                                                                                                           temperature
                                                                                                                            15⁰C~25⁰C
                                                                                                                                                 Description
                                                                                                                                                 Plant Genomic DNA Maxi Kits provide a fast and simple method to isolate total DNA (genomic DNA,
                                                                                                                                                 mitochondrial and chloroplast) from plant tissue and cells. Samples are homogenized under liquid nitrogen
                                                                                                                                                 followed by lysis buffer incubation. The lysate is treated with RNase A to degrade RNA and spun through a filter
                                                                                                                                                 column to remove cell debris and salt in the solution. In the presence of binding buffer and chaotropic salt, the
       Cat.No. YGPM10                               Cat.No. YGPM25                                                                               genomic DNA in the lysate binds to the glass fiber matrix in the spin column. The contaminants are washed with
                                                                                                                                                 an ethanol based wash buffer and the purified genomic DNA is eluted by low salt elution buffer or water. The
       10 maxi preps / kit                          25 Maxi preps / kit                                                                          purified genomic DNA is ready for PCR, real-time PCR, Southern blotting and RFLP.
       GP1 Buffer: 50 ml                             GP1 Buffer: 125 ml
       GPX1 Buffer: 50 ml                            GPX1 Buffer: 125 ml                                                                           Protocol Modifications (Duo 2 Buffer System)
                                                                                                                                                 Plant species are extremely diverse in their metabolic components. Large amounts of polysaccharides,
       GP2 Buffer: 15 ml                             GP2 Buffer: 30 ml                                                                             carbohydrates, lipids, polyphenols and proteins may be distributed throughout the plant tissue. These
       GP3 Buffer(concentrated): 30 ml *             GP3 Buffer(concentrated): 70 ml *                                                             compounds often interfere with DNA binding and extraction. This characteristic of plants means we offer two
       Wash Buffer (concentrated): 50 ml **          Wash Buffer (concentrated): 50 ml x 2 **                                                      lysis buffers for optimum performance according to plant sample type.
       Elution Buffer: 30 ml                         Elution Buffer: 60 ml
       RNase A(10 mg/ml): 550 μl                    RNase A(10 mg/ml): 650 μl x 2
                                                                                                                                                 GP1: The standard protocol utilizes GP1 buffer for lysis of plant samples. This buffer system is suitable for most
                                                                                                                                                 common plant species, ensuring high quality and high yields of DNA.
       Filter Column: 10 pcs                        Filter Column: 25 pcs
       GD-Maxi Column: 10 pcs                       GD-Maxi Column: 25 pcs                                                                       GPX1: An alternative buffer, GPX1, is also included with this kit. The detergent present in this buffer is more
                                                                                                                                                 effective in dispersing plant samples with large amounts of polysaccharide. For the majority of plant species both
       Sample: 1 g of plant tissue                                                                                                               buffers will give similar results. The researcher may try one buffer system first or both in parallel.
       Yield: 50-300 μg                                                                                                                          Quality Control
                                                                                                                                                 The quality of the Plant Genomic DNA kits are tested on a lot-to-lot basis. The kits are tested by isolation of
       Operation time: < 80 min.
                                                                                                                                                 genomic DNA from 500 mg of young leaves.
                                                                                                                                                 Caution
     * Add two times volume of Isopropanol to GP3 buffer: 60 ml/ 140 ml                                                                           The above components contain irritant agents. During the extraction process always wear a lab coat, disposable
    ** Add four times volume of Ethanol to Wash Buffer: 200 ml / 400 ml                                                                           gloves and protective goggles.


1                                                                                                                                                                                                                                                                                    2

                                                                                                                                                                                                           Genomic DNA Extraction Kit [Maxi](Plant)
      Maxi Protocol                                                                                                                              Wash
                                                                                                                                                 14. Add 10 ml of Wash Buffer (ethanol added) into the column.
      Tissue Dissociation                                                                                                                        15. Centrifuge at 4,000 X g for 3 minutes.
      1. Cut off 0.5 g (up to 1g) of fresh or frozen plant tissue or 50 mg (up to 100 mg) of dried sample.                                        16. Discard the flow-through and place the GD-Maxi Column back in the collection tube.
      2. Grind the sample under liquid nitrogen to a fine powder. Transfer it into a 15 ml centrifuge tube (not provided).                        17. Repeat the Wash step again.
         It is possible to render some plant samples without the aid of liquid nitrogen.                                                         18. Discard the flow-through and place the GD-Maxi Column back in the collection tube.
                                                                                                                                                 19. Centrifuge at 4,000 X g for 10 minutes to dry the column matrix.
      Lysis
      2. Add 4 ml GP1 Buffer (or GPX1 Buffer) and 50 μl RNase A (10 mg/ ml) into the sample tube and mix by vortexing.
         (Do not mix GP1 buffer and RNase A before use.)
                                                                                                                                                 DNA Elution
                                                                                                                                                 Standard elution volume is 1 ml. If less sample is used, reduce the elution volume (200-500 μl) to increase DNA concentration. If
      3. Incubate at 65⁰C for 20 minutes. During incubation, invert the tube every 5 minutes.At the same time, preheat required Elution
                                                                                                                                                 higher DNA yield is required, repeat the DNA Elution step to increase DNA recovery by bringing the total elution volume to about
         Buffer (2 ml per sample) at 65⁰C.
                                                                                                                                                 2 ml.
      4. Add 1 ml GP2 Buffer and mix by vortexing.
      5. Incubate at ice for 5 minutes.
                                                                                                                                                 20. Transfer dried GD-Maxi into a clean 50 ml centrifuge tube (not provided).
      6. Place a Filter Column in a 50 ml centrifuge tube (not provided).
                                                                                                                                                 21. Add 1 ml of preheated Elution Buffer into the center of the column matrix.
      7. Apply the mixture from previous step to the Filter Column. Centrifuge at 4,000 X g for 5 minutes.
                                                                                                                                                 22. Stand for 5 minutes until Elution Buffer absorbed by the matrix.
      8. Discard the Filter Column and carefully transfer clarified supernatant in collection tube to a new 50 ml centrifuge tube
                                                                                                                                                 23. Centrifuge at 4,000 X g for 3 minutes to elute purified DNA.
         (not provided).

      DNA Binding
      9. Add 1.5 volumes of GP3 Buffer (isopropanol already added) to the cleared lysate and mix immediately by vortexing for 10
         seconds. For example, add 7.5 ml GP3 Buffer to 5 ml of lysate.
      10. Place a GD-Maxi Column in a 50 ml centrifuge tube.
      11. Apply the mixture (including any precipitate) from previous step to the GD-Maxi Column.
      12. Centrifuge at 4,000 X g for 5 minutes.
      13. Discard the flow-through and place the GD-Maxi Column back in the collection t ube.

3                                                                                                                                                                                                                                                                                    4

                                                                                                                                                                                                Genomic DNA Extraction Kit [Midi/Maxi](Plant)




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