From Kyle Harris and Alison Nairn: 4/12/07 Total RNA isolation from cell pellets General considerations when working with RNA: - wear gloves at all times - use RNase-free tips and tubes for any samples (autoclaved and not touched without gloves) - use RNase-free solutions (either Molecular Grade Water [Mediatech in supply center] or order DEPC-treated solutions from Ambion) - Trizol contains denaturants and is hazardous (similar to working with Phenol) Protocol: 1. Add 2 ml Trizol (Invitrogen)(in 4 ° C fridge) to 2.5 x 10 6 cells (frozen pellet or fresh cells). 2. Homogenize with polytron or resuspend cell pellet using a 3 ml syringe and 21-guage needle, 3 cycles through needle. 3. Switch to a 22-gauge needle and process 3 times. 4. Incubate homogenized samples for 5 min at room temp. 5. Add 200 μ l (1/10 th volume) chloroform/isoamyl alcohol (49:1, v/v), cover tubes tightly and shake vigorously for 15 sec. 6.
This is the end of the preview.
access the rest of the document.