{[ promptMessage ]}

Bookmark it

{[ promptMessage ]}


RNAisolationProtocol - From Kyle Harris and Alison Nairn...

Info iconThis preview shows page 1. Sign up to view the full content.

View Full Document Right Arrow Icon
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.
Background image of page 1
This is the end of the preview. Sign up to access the rest of the document.

{[ snackBarMessage ]}

Ask a homework question - tutors are online