Zymo rna clean concentrator 5

Frozen cell preparations (100,000 cells in 750 μL of Trizol-LS) were thawed completely at room temperature. 800 μL of the sample was then transferred to pre-spun Phase Lock Gel-Heavy Tubes (Quantabio #2302830) and incubated at room temperature for 5 min. Next, 160 μL of chloroform was added to each tube, vigorously shaken for 15 s, and incubated at room temperature for 2 min. Samples were then centrifuged at 12,000xg for 15 min at 4°C. Following centrifugation, the aqueous phase (∼400 μL) was transferred to fresh 1.7 mL tube. 1 volume of 100% ethanol was added to the recovered aqueous phase and mixed by pipetting. The sample was then transferred to a Zymo RNA Clean & Concentrator-5 (Zymo Research #R1015) spin column and centrifuged at 16,000xg for 30 s. Flow through was discarded at each step prior to elution. 400 μL of RNA Prep Buffer was added to the column and centrifuged at 16,000xg for 30 s. Then the column was washed twice with RNA Wash Buffer. First, with 700 μL at 16,000xg for 30 s and then with 400 μL at 16,000xg for 2 min to dry column. RNA was eluted from column with 10 μL of DNase/RNase-Free water by centrifugation at 16,000xg for 30 s.