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<br> (1) Blocking: 5% whole milk incubate for 1h | <br> (1) Blocking: 5% whole milk incubate for 1h | ||
<br> (2) Incubate with diluted primary antibody | <br> (2) Incubate with diluted primary antibody | ||
− | <br> (3) Wash membrane: TBST | + | <br> (3) Wash membrane: TBST 15 mins *4 |
<br> (4) Incubate with diluted secondary antibody | <br> (4) Incubate with diluted secondary antibody | ||
− | <br> (5) Wash membrane: TBST | + | <br> (5) Wash membrane: TBST 15 mins *4 |
− | <br> (6) Add Chemiluminescene substrate, incubate in dark for | + | <br> (6) Add Chemiluminescene substrate, incubate in dark for 5 mins |
<br> (7) Exposure | <br> (7) Exposure | ||
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plasmids.) | plasmids.) | ||
− | <br> 2. Spin the bacterial culture at 6,000 rpm for 10 | + | <br> 2. Spin the bacterial culture at 6,000 rpm for 10 mins at 4 C. 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 | + | <br> 7. Spin the lysate at 10,000 rpm for 10 mins at 4 C. |
<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 | + | for 10 mins at room temperature. |
− | <br> 9. Spin at 10,000 rpm for 10 | + | <br> 9. Spin at 10,000 rpm for 10 mins at 4 C. 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 | + | <br> 11. Add 200 ul of 8M LiCl. Mix well, and then spin the solution at 14,000 rpm for 5 mins at 4 C. |
<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 | + | well, and then spin the solution at 14,000 rpm for 5 mins at 4 C. |
<br> 13. Discard the supernatant. Rince the pellet and the wall of the tube with 500 ul of cold 70% ethanol. Discard | <br> 13. Discard the supernatant. Rince the pellet and the wall of the tube with 500 ul of cold 70% ethanol. Discard | ||
the fluid. | the fluid. | ||
− | <br> 14. Add to the pellet 400 ul of TE containing DNase-free RNase A (20 ug/ml). Incubate the tube for 30 | + | <br> 14. Add to the pellet 400 ul of TE containing DNase-free RNase A (20 ug/ml). Incubate the tube for 30 mins at |
37 C. | 37 C. | ||
− | <br> 15. After 30 | + | <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 37 C for further 30 | + | 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 | ||
to PEG8000, and works well.) | to PEG8000, and works well.) | ||
− | <br> 17. Spin at 14,000 rpm for 5 | + | <br> 17. Spin at 14,000 rpm for 5 mins. Discard the supernatant. Rinse the pellet with 300 ul of cold 70% ethanol. |
Discar the fluid. Dissolve the pellet in 400 ul of TE. (Optional: Repeat PEG precipitation once more. This is | Discar the fluid. Dissolve the pellet in 400 ul of TE. (Optional: Repeat PEG precipitation once more. This is | ||
recommended for preparing dephosphorylated linear vector since trace amount of short RNA in the vector preparation | recommended for preparing dephosphorylated linear vector since trace amount of short RNA in the vector preparation | ||
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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 | + | for 5 mins at 4 C. 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. Cycling Conditions: | <br> 2. Cycling Conditions: | ||
− | <br> Step 1: 95℃, | + | <br> Step 1: 95℃, 5mins |
<br> Step 2: 95℃, 30s | <br> Step 2: 95℃, 30s | ||
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<br> alternative for tubes: vortex 1 min | <br> alternative for tubes: vortex 1 min | ||
− | <br> alternative for tissue: grind 1 g tissue in liquid nitrogen in a motar and pestle, put tissue into plastic screw-cap centrifuge tube + 15 ml TRIzol reagent, incubate samples for 5 | + | <br> alternative for tissue: grind 1 g tissue in liquid nitrogen in a motar and pestle, put tissue into plastic screw-cap centrifuge tube + 15 ml TRIzol reagent, incubate samples for 5 mins at room temp or 60° C (scaled up as needed) |
− | <br> (4) ( | + | <br> (4) (5 mins at RT for complete dissociation of nucleoprotein complexes) |
<br>RNA is stable in trizol which deactivates RNases. You can take a break at this point keeping the sample | <br>RNA is stable in trizol which deactivates RNases. You can take a break at this point keeping the sample | ||
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<h4> <br>2.phase separation</h4> | <h4> <br>2.phase separation</h4> | ||
<h4 style="padding-left:30px;"> | <h4 style="padding-left:30px;"> | ||
− | <br>15-45 | + | <br>15-45 mins depending on number of samples and whether an additional chloroform wash is necessary |
<br> (1) add chloroform (1/5 volume of trizol; e.g. 0.2ml to 1ml) | <br> (1) add chloroform (1/5 volume of trizol; e.g. 0.2ml to 1ml) | ||
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<br> (2) shake for 15 sec (Eccles protocol: do not vortex) | <br> (2) shake for 15 sec (Eccles protocol: do not vortex) | ||
− | <br> (3) incubate 2-5 | + | <br> (3) incubate 2-5 mins at RT |
− | <br> (4) spin max. 12000g, 5-15 | + | <br> (4) spin max. 12000g, 5-15 mins, 2-8°C |
<br> if centrifugation hasn't been sufficient the DNA-containing interphase will be cloud-like and poorly compacted | <br> if centrifugation hasn't been sufficient the DNA-containing interphase will be cloud-like and poorly compacted | ||
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<h4 > <br>3.RNA precipitation and wash</h4> | <h4 > <br>3.RNA precipitation and wash</h4> | ||
<h4 style="padding-left:30px;"> | <h4 style="padding-left:30px;"> | ||
− | <br>20-40 | + | <br>20-40 mins depending on number of samples |
<br>(1) add isopropanol (70% of aqueous phase or 1/2 trizol volume) | <br>(1) add isopropanol (70% of aqueous phase or 1/2 trizol volume) | ||
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<br>(2) 0.8 M sodium citrate or 1.2 M NaCl can be added | <br>(2) 0.8 M sodium citrate or 1.2 M NaCl can be added | ||
− | <br>(3) (incubate | + | <br>(3) (incubate 10 mins at RT) |
− | <br>(4) spin max g, 10-15 | + | <br>(4) spin max g, 10-15 mins, 4ºC |
<br>(5) remove supernatant | <br>(5) remove supernatant | ||
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<h4 > <br>4.RNA wash</h4> | <h4 > <br>4.RNA wash</h4> | ||
<h4 style="padding-left:30px;"> | <h4 style="padding-left:30px;"> | ||
− | <br> 15-30 | + | <br> 15-30 mins( depending on number of samples) |
<br>(1) wash pellet 70% EtOH (add & vortex briefly) | <br>(1) wash pellet 70% EtOH (add & vortex briefly) | ||
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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 | + | <br>(2) spin max g, 2-10 mins, 4ºC |
− | <br> (3) air-dry pellet for 5-10 | + | <br> (3) air-dry pellet for 5-10 mins Do not overdry the pellet or you won''t be able to redissolve it. |
<br> optional add RNase inhibitor | <br> optional add RNase inhibitor | ||
− | <br> (4) incubate at 55-60 C° for 10 | + | <br> (4) incubate at 55-60 C° for 10 mins if hard to redissolve |
<br>(5) transfer to eppendorf tube | <br>(5) transfer to eppendorf tube | ||
− | <br> (6) spin 4° C, 5 | + | <br> (6) spin 4° C, 5 mins (to pellet undissolved material) |
</h4> | </h4> | ||
<h4 ><br>5.redissolving of RNA</h4> | <h4 ><br>5.redissolving of RNA</h4> | ||
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<br> (1) dissolve pellet in 50-100 µl filtered or DEPC H2O (note: DEPC inhibits RT reaction) | <br> (1) dissolve pellet in 50-100 µl filtered or DEPC H2O (note: DEPC inhibits RT reaction) | ||
− | <br>(2) alternatively, 0.5% SDS, pipetting up and down, heat to 55-60°C for 10 | + | <br>(2) alternatively, 0.5% SDS, pipetting up and down, heat to 55-60°C for 10 mins |
</h4> | </h4> | ||
</div> | </div> |
Revision as of 11:52, 1 November 2017