YuchuanCPU (Talk | contribs) |
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using a pipette or cell scraper. | using a pipette or cell scraper. | ||
− | <br> 4. Centrifuge at | + | <br> 4. Centrifuge at 4℃ for 15 mins at 15000 rpm to pellet insoluble material. |
<br> 5. Carefully remove supernatant to a fresh tube. | <br> 5. Carefully remove supernatant to a fresh tube. | ||
− | <br> 6. Add 1 μg of primary antibody and incubate with gentle mixing for 2-16 hours at | + | <br> 6. Add 1 μg of primary antibody and incubate with gentle mixing for 2-16 hours at 4℃. |
<br> 7. Wash 20 ml of Protein G-sepharose in 1ml of lysis buffer | <br> 7. Wash 20 ml of Protein G-sepharose in 1ml of lysis buffer | ||
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<br> 8. Resuspend to 100 μl and add to lysate containing antibody. | <br> 8. Resuspend to 100 μl and add to lysate containing antibody. | ||
− | <br> 9. Incubate 1 hour with gentle mixing at | + | <br> 9. Incubate 1 hour with gentle mixing at 4℃. |
− | <br> 10. Wash complexes 3 times in lysis buffer + PIC at | + | <br> 10. Wash complexes 3 times in lysis buffer + PIC at 4℃. |
<br> 11. Boil in 50 μl SDS sample buffer prior to loading on gel | <br> 11. Boil in 50 μl SDS sample buffer prior to loading on gel | ||
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plasmids.) | plasmids.) | ||
− | <br> 2. Spin the bacterial culture at 6,000 rpm for 10 mins at | + | <br> 2. Spin the bacterial culture at 6,000 rpm for 10 mins at 4℃. Discard the supernatant. |
<br> 3. (0ptional) Suspend the cell in about 20 ml of deionized water. Spin again. Discard the supernatant. Remove | <br> 3. (0ptional) Suspend the cell in about 20 ml of deionized water. Spin again. Discard the supernatant. Remove | ||
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<br> 6. Add 7.5 ml of Solution III. Mix well as above. | <br> 6. Add 7.5 ml of Solution III. Mix well as above. | ||
− | <br> 7. Spin the lysate at 10,000 rpm for 10 mins at | + | <br> 7. Spin the lysate at 10,000 rpm for 10 mins at 4℃. |
<br> 8. Transfer the supernatant into a new bottle. Add about 15 ml of isopropanol, mix well, and store the bottle | <br> 8. Transfer the supernatant into a new bottle. Add about 15 ml of isopropanol, mix well, and store the bottle | ||
for 10 mins at room temperature. | for 10 mins at room temperature. | ||
− | <br> 9. Spin at 10,000 rpm for 10 mins at | + | <br> 9. Spin at 10,000 rpm for 10 mins at 4℃. Discard the supernatant. Remove all of the fluid using pipetman. |
<br> 10. Dissolve the pellet in 600 ul of TE. Transfer the solution into a microfuge tube. | <br> 10. Dissolve the pellet in 600 ul of TE. Transfer the solution into a microfuge tube. | ||
− | <br> 11. Add 200 ul of 8M LiCl. Mix well, and then spin the solution at 14,000 rpm for 5 mins at | + | <br> 11. Add 200 ul of 8M LiCl. Mix well, and then spin the solution at 14,000 rpm for 5 mins at 4℃. |
<br> 12. Transfer the supernatant containing plasmid DNA to a new microfuge tube. Add 600 ul of isopropanol. Mix | <br> 12. Transfer the supernatant containing plasmid DNA to a new microfuge tube. Add 600 ul of isopropanol. Mix | ||
− | well, and then spin the solution at 14,000 rpm for 5 mins at | + | well, and then spin the solution at 14,000 rpm for 5 mins at 4℃. |
<br> 13. Discard the supernatant. Rinse the pellet and the wall of the tube with 500 ul of cold 70% ethanol. Discard | <br> 13. Discard the supernatant. Rinse the pellet and the wall of the tube with 500 ul of cold 70% ethanol. Discard | ||
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<br> 15. After 30 mins, carefully check the content of the tube. If nucleic acid pellet is visible at the bottom of | <br> 15. After 30 mins, carefully check the content of the tube. If nucleic acid pellet is visible at the bottom of | ||
− | the tube, vortex well to dissolve the pellet. Incubate the tube at | + | the tube, vortex well to dissolve the pellet. Incubate the tube at 37°C for further 30 mins. |
<br> 16. Add 240 ul of 2M NaCl, 20% PEG8000. (PEG6000 supplied from Japanese suppliers is essentially equivalent | <br> 16. Add 240 ul of 2M NaCl, 20% PEG8000. (PEG6000 supplied from Japanese suppliers is essentially equivalent | ||
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<br> 19. Add 0.1 volume of 3 M sodium acetate and 3 volume of ethanol into the plasmid solution. Spin at 14,000 rpm | <br> 19. Add 0.1 volume of 3 M sodium acetate and 3 volume of ethanol into the plasmid solution. Spin at 14,000 rpm | ||
− | for 5 mins at | + | for 5 mins at 4℃. Discard the supernatant. Rinse the pellet with 200 - 500 ul of 70% ethanol. |
<br> 20. Store the open tube on the bench until the visible traces of ethanol have evaporated. | <br> 20. Store the open tube on the bench until the visible traces of ethanol have evaporated. | ||
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<br>2.Replace the old medium with 6 ml of fresh culture medium. Add 60 µl of 0.6 M sodium butyrate. Return to the incubator. | <br>2.Replace the old medium with 6 ml of fresh culture medium. Add 60 µl of 0.6 M sodium butyrate. Return to the incubator. | ||
− | <br>3.After 48 hours of culture, collect the supernatant and freeze it at - | + | <br>3.After 48 hours of culture, collect the supernatant and freeze it at -80℃ or proceed to the concentration step. |
</h4> | </h4> | ||
<h4 style="font-weight:600"> | <h4 style="font-weight:600"> | ||
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<br> 3.Transfer the supernatant to autoclaved polyallomer tubes. Concentrate the supernatant by ultracentrifugation | <br> 3.Transfer the supernatant to autoclaved polyallomer tubes. Concentrate the supernatant by ultracentrifugation | ||
− | for 1.5 hours at | + | for 1.5 hours at 4℃ in a Beckman SW 28 swinging bucket rotor at 24,500 rpm. |
<br> 4.Remove the supernatant and resuspend the pellet in an appropriate amount of culture medium, e.g., | <br> 4.Remove the supernatant and resuspend the pellet in an appropriate amount of culture medium, e.g., | ||
300 µl for 30 ml of original supernatant if a 100-fold concentration is desired. | 300 µl for 30 ml of original supernatant if a 100-fold concentration is desired. | ||
− | <br> 5.Divide the concentrated vector into 10-50-µl aliquots and store at - | + | <br> 5.Divide the concentrated vector into 10-50-µl aliquots and store at -80℃ until use. |
</h4> | </h4> | ||
<h4 style="font-weight:600"> | <h4 style="font-weight:600"> | ||
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<br>(3) (incubate 10 mins at RT) | <br>(3) (incubate 10 mins at RT) | ||
− | <br>(4) spin max g, 10-15 mins, | + | <br>(4) spin max g, 10-15 mins, 4℃ |
<br>(5) remove supernatant | <br>(5) remove supernatant | ||
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<br>some prefer to wash the pellot more than once with 70% ethanol | <br>some prefer to wash the pellot more than once with 70% ethanol | ||
− | <br>(2) spin max g, 2-10 mins, | + | <br>(2) spin max g, 2-10 mins, 4℃ |
<br> (3) air-dry pellet for 5-10 mins Do not overdry the pellet or you won''t be able to redissolve it. | <br> (3) air-dry pellet for 5-10 mins Do not overdry the pellet or you won''t be able to redissolve it. | ||
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<br>(5) transfer to eppendorf tube | <br>(5) transfer to eppendorf tube | ||
− | <br> (6) spin | + | <br> (6) spin 4℃, 5 mins (to pellet undissolved material) |
</h4> | </h4> | ||
<h4 ><br>5.redissolving of RNA</h4> | <h4 ><br>5.redissolving of RNA</h4> |
Latest revision as of 15:19, 1 November 2017