To COS-M6 cells grow in 6-well plates, 2ml media total
To CV1PD 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 (4oC is OK). Be very gentle when washing the cells as transfected cells tend to come 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. To COS-M6 cells, they can be washed off simply by pipetting 20 times per well. To CV1-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 100ul ice cold TD by pipetting up and down 10 times. Add 100ul 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 250ul 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: To cytoplasmic fractions, add 0.9ml 1.2xTRIzol reagent. Vortex to mix completely, sit at RT for 5 minutes.
8: Add 200ul chloroform:IAA 200ul per 1ml TRIzol. Vortex at scale 6 for 1 min.
9: 4oC spin 20min, less than 12000 g. The RNA should exist in supernatant exclusively
10. Transfer supernatant 3x200ul = 600 ul to a new tube carefully. The total volume of the supernatant should around 650-700ul, 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: 4oC 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 50ul 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
Na2HPO4 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 60oC 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 60oC 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)
Preparation of total cel