HMW RNA extraction from E.coli

High molecular weight (HMW) RNA ( > 200 nt) of E.coli (BL21) DE3 was extracted using MiniBEST Universal RNA Extraction Kit. E. coli strain BL21 (DE3) was cultured in LB broth and shaken overnight (230 rpm) at 16 °C. The cells were pelleted by centrifugation at 13,800 × g for 20 min at 4 °C and washed with 1× PBS water to remove residual LB broth. A total of 350 μL lysis Buffer RL was added to the collected cells. The lysate was transferred to a gDNA Eraser Spin Column and centrifuged at 13,800 × g for 1 min at 20 °C to remove the gDNA. The filtrate was added with isopycnic 70% ethanol and mixed thoroughly. The mixture was transferred to RNA Spin Column and centrifuged at 13800 × g for 1 min at 20 °C. The RNA Spin Column was added with 500 μL Buffer RWA and centrifuged at 13,800 × g for 30 s at 20 °C. The filtrate was discarded. The RNA Spin Column was added with 600 μL buffer RWB and centrifuged at 13800 × g for 3 min at 20 °C. The RNA Spin Column was placed onto 1.5 mL RNase Free Collection Tube and added with 30–200 μL RNase free water. After 5 min, HMW RNA was eluted by centrifugation at 13,800 × g for 2 min at 20 °C. The concentration was measurement using nanodrop and the desired fraction was determined using 1% agarose gel electrophoresis. Finally, HMW RNA was stored at −80 °C for subsequent electrophysiology measurements. Tips and tubes used were RNase-free.