RNA extraction via Pellet and LiCl purification

1) Remove Trizol-preserved samples and let them thaw

2) Extract 1.5 ml from sample into a 2.0 ml eppendorf tube (make triplicate)
3) Add 300 l of chloroform to sample and shake vigourously for 15 seconds. Let sample sit
for 2 minutes.
4) Spin in a centrifuge at 12,000 g for 15 minutes at 4
o
C.
5) Extract upper layer (~400-600 l) and put into a fresh 2.0 ml eppendorf tube. Discard old
tube into a Trizol waste bin.
6) Add 375 l of 100% isopropyl alcohol to sample.
7) Add 375 l of High Salt Solution (vortex before use)
8) Let sample sit for 10 minutes at RT.
9) Centrifuge at 12,000 g for 10 minutes at 4
o
C.
10) Pour supernatant into waste beaker.
11) Add 1.5 ml of cold 75% EtOH from -20
o
C and vortex to dislodge pellet.
12) Spin at 8,000 rpm for 5 minutes at 4
o
C.
13) Empty tube and let it air dry for ~15 minutes
14) Add 50 l of RNase-free water to sample and dissolve pellet
15) Combine aliquots into a 0.6 ml eppendorf tube (total volume should be 150 l).
16) Add 150 l of 13.3 M LiCl to 0.6 ml tube and place in -20
o
C for at least 30 minutes
17) Centrifuge at 16,000 g for 20 minutes at RT.
18) Add 200 l of 80% EtOH to tube and empty (DO NOT SHAKE)
19) Add 200 l of 80% EtOH to tube and quick spin (do not centrifuge).
20) Empty tube and let it air dry for 15 minutes
21) Dissolve pellet in 20 l of RNase-free water.
22) Quantify sample on Nanodrop spectrophotometer to determine quantity and purity of
sample.