T℃OS-M6 cells grow in 6-well plates, 2ml media total T℃V1PD cells grow in 100mm plates. 1: Aspirate off the media from the cells and was the cell monolayers with 2x5ml (To 100mm dish) or 2x1ml (to 6-well plates) ice cold TD (4o C is OK). Be very gentle when washing the cells as transfected cells tend t℃ome off the dish easily. You had better transfect 9 dishes or 18 wells at a same time, so that you will have 18 Eppendorff tubes that can be spun down in one centrifuge. 2: Add 1ml ice cold TD to the well. T℃OS-M6 cells, they can be washed off simply by pipetting 20 times per well. T℃V1-PD cells, they should be scraped by rubber policeman. Handle the rubber policeman at the first 1/3 so that you will not spill the solution. Transfer the cells to a 1.5ml Eppendorff tube. Put each Eppendorff tube on ice until donw with all samples. 3: Spin cells at RT for 30sec at maxi speed From now on, keep tubes on ice as much as possible 4: Remove supernatant with aspirator. Resuspend the cells with 100μl ice cold TD by pipetting up and down 10 times. Add 100μl VRC/1%NP-40/TD. Vortex 5 second and put on ice. VRC/1%NP-40/TD: 2.4 ml 1%NP-40/TD 125ul VRC 4.8 ml 1%NP-40/TD 250μl VRC 5: Keep on ice for 5 min, vortex 5 seconds again. Quick spin 30 seconds at maxi speed at RT. 6: Transfer cytoplasmic fraction to a new tube, put nuclear samples on ice now. 7: T℃ytoplasmic fractions, add 0.9ml 1.2xTRIzol reagent. Vortex to mix completely, sit at RT for 5 minutes. 8: Add 200μl chloroform:IAA 200μl per 1ml TRIzol. Vortex at scale 6 for 1 min. 9: 4o C spin 20min, less than 12000 g. The RNA should exist in supernatant exclusively 10. Transfer supernatant 3x200μl = 600 ul to a new tube carefully. The total volume of the supernatant should around 650-700μl, no need to transfer them all. 11: Add 0.25ml isopropanol 0.25ml RNA precipitation solution Mix by vortex and sit at RT for 10 min 12: 4o C spin 10min 13: Aspirate off supernatant, wash with 70% EtOH, spin 5min at RT 14: Dry without heat in speed vac for 5 to 10 min 15: Resuspend in 50μl TE completely. |
To nuclear fractions: 16: Resuspend nuclear samples in 200 ul 0.5%NP-40/TD by pipetting 10 times. Vortex 5 seconds and put on ice for 5 minutes. 16: Spin 30 seconds at RT, maxi speed. 17: Aspirate off supernatant and resuspend samples in 200 ul 0.5%NP-40/TD by pipetting 10 times again. 18: Follow the steps from 7 to 15 again.
Solutions: TD: 2L NaCl 16.0g KCl 0.76g Na2 HPO4 0.20g Tris-Cl 6g Water to 2 L, pH 7.4-7.5 1xTRIzol: 200ml Final Add Guanidine thiocyanate 0.8 M FW 118.2 18.912 g Ammonium thiocyanate 0.4 M FW 76.12 6.09 g Sodium Acetate 0.1 M, pH 5 Stock 1M 20 ml Glycerol 5% 10ml
Add water to 100ml Mix well Filter Add phenol 76 ml and mix. Phenol can be solved in 60℃ water bath. The final concentration of phenol is 38%. Add water to 200 ml total volume. pH ~ 4.5. Wrap with aluminum foil. 1.2xTRIzol: 200ml Final Add Guanidine thiocyanate 0.8 M x 1.2 = 0.96 M 22.694 g Ammonium thiocyanate 0.4 M x 1.2 = 0.48 M 7.308 g Sodium Acetate 0.1 M x 1.2 = 0.12 M, pH 5 Stock 1M 24 ml Glycerol 5% x 1.2 = 6% 12ml
Add water to 100ml Mix well Filter Add phenol 76 ml x 1.2 = 91.2 and mix. Phenol can be solved in 60o C water bath. The final concentration of phenol is 38% x 1.2 = 45.6%. Add water to 200 ml total volume. pH ~ 4.5. Wrap with aluminum foil. RNA precipitation solution: 200 ml NaCl 1.2 M 14.026 g FW 58.44 Sodium Citrate 0.8M 47.056 g FW 294.10 Autoclaved (can't be filtered through the membrane) |
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