446/546 handout RNA extraction
Today you will be extracting RNA from Physella acuta snails.
RNA extraction
Note: It is very important to wear gloves at all times. You’ll work with some hazardous
chemicals, and your skin contains RNAses that can degrade the RNA that you are trying to
extract.
1. Apply RNASEZap spray to your bench and the pestle that will be used to disrupt the
snail tissues
2. Add 250uL of Trizol reagent to a 1.7 ml Eppendorf tube
3. Clean the snail with a kim wipe
4. Use tweezers to place the snail in the 1.7 ml Eppendorf tube containing Trizol
5 Grind with a pestle, after the tissue is properly ground, add 750uL Trizol and mix 3 times
6 Incubate for 3 min room temp (rT)
7 All to start this step at the same time: Spin down for 10 min at max speed
8 Carefully transfer aqueous layer to a new snap cap.
9 Add 200 uL of chloroform for each 1mL of Trizol
10 Shake vigorously and incubate for 3 min rT
11 All to start this step at the same time Centrifuge 15 min at max speed
12 Carefully transfer aqueous layer to new snap cap
13 Add 500 uL of isopropanol for each 1 mL of Trizol, mix well by inverting.
14 All to start this step at the same time Incubate at rT for 10 min
15 Spin down for 10 min at max speed
16 Decant supernatant; you should be able to see the pellet at this step
17 Wash with 1mL of 75% ethanol and vortex
18 Spin down for 5 min at max speed
19 Decant supernatant, remove drops,
20 All to start this step at the same time air dry for 5 min
21 Resuspend in 50 uL of EB buffer or water
Turbo DNA-free
1. Add 6uL of 10X buffer/1 uL enzyme master mix, 3 uL mQ and mix (vortex)
2. Incubate at 37°/30min
3. Add 6 uL of inactivating reagent, vortex and incubate for 2 minutes at rt
4. Spin down at max speed for 1 min
5. Carefully pipet supernatant (~50 uL)
6. This is your sample, proceed to bioanalyzer