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    DNA/RNA/Protein Purification from Cultured Cells Using SQ DNA/RNA/Protein Cell Kit (1-2 x 106 cells)

    互联网

    1103

    实验原理

     

    If using the SQ DNA/RNA/Protein for the first time, please read this booklet to become familiar with the procedure and its various modifications. Samples are first lysed in a specially formulated buffer. The protein is precipitated by adding Protein Precipitation Reagent (PPR). After removal of the protein, the supernatant is mixed with 1 volume of isopropanol to precipitate the RNA. After the centrifugation to pellet the RNA, the supernatant is transfer to a new tube and mixed with DPR Buffer to precipitate DNA. RNA and DNA pellet are washed with 70% ethanol and dissolved with water or low ionic strength buffer. Purified DNA, RNA and protein can be directly used in downstream applications without the need for further purification.

    实验试剂

     

    Regents supplied by User

    1. Absolute ethanol

    2. 70% ethanol

    3. 100% Isopropanol

    4. Ice

    实验设备

     

    Equipments supplied by User

    1. Centrifuge with temperature control

    2. Table top centrifuge capable at least 13,000 x g

    3. 1.5 or 2 ml Nuclease-Free centrifuge tubes

    4. 0.45 μm filter unit

    实验步骤

     

    Before starting:

    1. Fresh or flash-frozen cultured cells can be used in this protocol. Collect suspended cells and place on ice until use. Determine the cell number by using a hemacytometer or other cell counter.

    2. Preheat the water bath to 65°C

    3. Water bath or heating block preset at 37°C

    4. Pre-set the centrifuge at 4°C for RNA isolation.

    5. Frozen cells should be thawed quickly using a 37°C water bath with gently agitation and place on ice until use.

    6. Warm up the Cell Lysis Buffer at 37°C water bath

    Procedure

    1. Harvest and lyse the cell:

       1) Cells Grown in Suspension

    Pellet cells by centrifugation in a 1.5 ml tube. Lyse cells in Cell Lysis Buffer by repetitive pipetting. Alternately, vortex the tube at maximum speed for 1 minute to lysis the cell. Use 300 μL of the reagent per 2 x 106 of cultured cells.

       2) Cells Grown in Monolayer

    Lyse cells directly in a culture dish by adding 300 μL of Cell Lysis Buffer per 2 x 106 of cultured cells directly into each well of multiwell cell culture plate or flask, and lyse the cell by passing the cell lysate several times through a blue pipette tip. Alternately, shake the plate on a orbital shaker at maximum speed for 1 minute to lysis the cells.

    2. Homogenize the samples:

    Cultured cells can be effectively homogenized by pipetting up and down or vortexing after addition of Cell lysis buffer.

    3. Place the tube containing the homogenates on the bench at room temperature for 2 minutes.

    4. Add 100 μL of Protein Precipitation Reagent (1/3 volume of Cells lysis Buffer) to each sample, mix throughly by votexing at maximum speed for 30 seconds.

    5. Incubate on ice for 10 minutes.

    6. Centrifuge at maximum speed ($13,000 x g) at 4°C for 10 minutes.

    7. Transfer cleared supernatant (-400 μL) into new 1.5 ml centrifuge tube for RNA/DNA isolation. Remove any liquid from the original tube contains protein precipitate by invert the plate on a absorbent paper. Keep the tube contains protein pellet for protein isolation start from step 26. For Total Nucleic acid isolation, proceed step 8-12. For RNA and DNA isolation, proceed step 13-25.

    Total Nucleic acid Isolation:

    8. Add equal volume (400 μL) of isopropanol. Mix throughly by vortexing for 30 seconds. Incubate the tube at room temperature for 5 minutes.

    9. Centrifuge at maximum speed ($13,000 x g) at 4°C for 10 minutes to precipitate Nucleic acid. Discord the supernatant.

    10. Wash the nucleic acid pellet by adding 400 μL of 70% ethanol into the tube. Vortex the tube for 30 seconds.

    11. Centrifuge at maximum speed ($13,000 x g) at 4°C for 3 minutes. Aspirate the supernatant and air dry the pellet by inverting the tube on a absorbent paper for 5-10 minutes.

    12. Add 50-100 μl of nuclease free water into the tube to dissolve the Nucleic acid pellet. Vortex the tube for 30 seconds. Incubate the tube on ice or 4°C for 30-45 minutes to completely re-hydrate the nucleic acid.

    For Total RNA Isolation:

    13. Add 200 μL Buffer DPR (½ volume of supernatant) into the cleared supernatant from step 7, vortex to mix well.

    14. Add equal volume (600 μL) of isopropanol. Mix throughly by vortexing. Incubate the tube at room temperature for 5 minutes.

    15. Centrifuge at maximum speed ($13,000 x g) at 4°C for 5 minutes to precipitate RNA. A bi-phase will be formed in the supernatant after the centrifugation.

    16. Transfer entire supernatant to a new 1.5 ml tube and keep the tube for DNA isolation starting at step 20.

    Note: It is critical to remove any liquid drop at this step to minimize the DNA contamination. For best result, briefly spin and collect drop from the tube, remove any drop by pipettor.

    17. Wash the RNA pellet by adding 600 μL of 70% ethanol into the tube. Vortex the tube for 30 seconds.

    18. Centrifuge at maximum speed ($13000 x g) at 4°C for 3 minutes. Aspirate the supernatant and air dry the RNA pellet by inverting the tube on a absorbent paper for 5-10 minutes

    19. Add 50-100μl of DEPC water into the tube to dissolve the RNA pellet. Vortex the tube for 30 seconds. Incubate the tube on ice or 4°C for 30-45 minutes to completely rehydrate the RNA.

    DNA Isolation:

    20. Add 1/6 volume of DPD Buffer (200 μL) and mix by vortexing.

    21. Centrifuge at maximum speed ($13,000 x g) for 5 minutes at room temperature to collect precipitated DNA. Carefully discard supernatant.

    22. Add 600 μL of 70% ethanol and mix throughly by vortexing for 30 seconds.

    23. Centrifuge at maximum speed ($13,000 x g) for 3 minutes at room temperature Carefully discard supernatant.

    24. Air dry the DNA pellet by inverting the tube on a absorbent paper for 5-10 minutes.

    25. Add 100ul EB Buffer to the tube and incubate at 65°C for 30 minutes to re-hydrate the DNA.

    Protein Isolation:

    26. Take the tube contains protein pellet from step 7.

    27. Add 1ml of 95% ethanol and vortex the tube for 30 seconds.

    28. Centrifuge at maximum speed for 2 minutes. Discard the supernatant. Air dry the protein pellet by inverting the tube on a absorbent paper for 5-10 minutes.

    29. Add 100-200uL protein loading dye (Laemmli Loading dye) to the tube to dissolve the protein pellet. If the protein will not be analyzed by SDS-PAGE, dissolve the protein in a buffer that compatible with downstream applications.

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