Preliminary Notes
1. Solutions are made without Tris, treated with 0.1% DEPC, then autoclaved, and Tris added.
2. Oligo dT-cellulose is recycled. Prior to use it is rinsed in 0.2 M NaOH once, then in binding buffer twice. After use it is rinsed in 0.2M NaOH once, then in elution buffer twice, then stored in elution buffer at 4¡C.
3. Column is washed with 0.2M NaOH, then several volumes of binding buffer.
Procedure.
1. From acid phenol method, resuspend in 5 ml STE/0.5M NaCl/Proteinase K 200 g/ml.
2. Add 0.25ml packed volume of oligo dT cellulose and mix on wheel for 30 min.
3. Spin oligodT down in benchtop at 2K 1' and wash with binding buffer (0.5 M NaCl, 10 mM TrisHCl pH 7.4, 1mM EDTA, 0.1% SDS). Remove supernatant and resuspend in binding buffer. Repeat twice.
4. Transfer to alkali treated econo-column. Wash with at least 6 volumes binding buffer.
5. Elute with 4 X elution buffer (1ml) into SW56 tube and ethanol precipitate by adding 100ul 5M NaCl and 2.5ml 100% ethanol. Leave at -20¡C overnight.
6. Spin 30K for 30 minutes. Pour off supernatant carefully and resuspend in 100ul 10mM EDTA. Reserve 5 ul for OD260/280 measurement. Ethanol precipitate remainder in 250ul ethanol, 10 ul 5M NaCl. Store at -70¡C.
Making 400 ml solutions, autoclave in 500 ml bottle
| H20 | 5M NaCl | 0.5M EDTA | 10% SDS | 1M Tris 7.4 | |
| add after | |||||
| autoclaving | |||||
| STE | 367.2 | 8 | 0.8 | 20 | 4 |
| Binding Buffer | 351.2 | 40 | 0.8 | 4 | 4 |
| Eluting Buffer | 391.9 | 0 | 0.8 | 4 | 4 |
| 10 mM EDTA | 392 | 8 | 0 | ||