| Total RNA preparation - Homogenise sample in minimum volume of TRIzol reagent to facilitate homogenisation,
add TRIzol reagent to total 1mL, 5 mins RT - Add 200µL chloroform, shake vigorously for 15 secs, 3 mins RT
- Spin 12000g at 4 degrees for 15 mins:
Phases separate into three: bottom red, phenol chloroform; middle interphase; upper clear colourless aqueous phase (ca. 60% vol) containing the RNA - Decant upper aqueous phase into fresh eppy
- Add 0.5mL isopropanol (RNA may visibly precipitate at this point),10 mins RT
- Spin 12000g at 4 degrees for 10 mins (should see RNA pellet), discard supe
- Wash with 1mL 70% ethanol (vortex and spin at 7000g at 4 degrees for 5 mins)
- Airdry RNA pellet until almost dry (completely dry pellet is harder to resuspend)
- Now resuspend in DEPC water, or continue with... Isolation of poly A+ RNA
- Resuspend in 1mL STE with proteinase K and SDS:
STE (0.1 M NaCl, 10 mM Tris 7.4, 1 mM EDTA) 50 ml | - | 1mL 5M NaCL stock | | - | 0.5mL 1M 10mM Tris 7.4 stock | | - | 0.1mL 0.5M EDTA stock | | - | 48.4mL dwater | STE with 200µg/mL proteinase K and 0.5% SDS: To 10mL STE add: | - | 100µL of 20mg/ml proteinase K stock | | - | 250µL of 20% SDS stock | - 10 mins RT (try to resuspend the pellet at this time) 1 hour at 60 degrees
- 10 mins RT (all RNA into solution by now)
- Make up to 0.5M NaCl (add 110µL 5M stock), spin out any debris
- Add ca. 200µL (packed volume) oligo(dT)-cellulose slurry in STE (0.5M NaCl)
- Wheel 10 mins RT
- Pulse spin and discard supe, resuspend in 1mL binding buffer
- Repeat binding buffer wash twice, transfer suspension to econocolumn
1mL washes with binding buffer
2mL wash with washing buffer - Elute with 2 x 200µL elution buffer
- Set aside 50µL for quantitation, adding to this 50µL of elution buffer
Read OD 260nm against elution buffer blank
To remaining 350µL, add 35 µL 5M NaCl and 800µL ethanol, store in freezer. |
