Guanidine hydrochloride guhcl

E. coli cells were harvested by centrifugation at 4500 rpm at 4°C for 10 minutes.
Approximately 10 8 E. coli cells, 10 7 yeast cells or 10 7 Chinese hamster ovary cells were suspended in pre-warmed 100 µl LB1 lysis reagent (4% SDS pH 7.5, 0.5 M NaCl) or LB2 lysis reagent (4% SDS pH 7.5, 0.5 M NaCl, 2% DMSO). For E. coli and mammalian cells, lysis was achieved by pipetting and incubating for 3 minutes.
However, for optimisation of RNA yield from yeast and E. coli cells expressing dsRNA, the suspended cells were heated for 4 minutes at 90°C and homogenised by pipetting. The lysate was then centrifuged for 4 minutes at 13,000 rpm and the supernatant transferred to a new 2 mL Eppendorf tube. 250 µL of 1.0 M guanidine hydrochloride (Gu-HCl) (Thermo Scientific), 40 µL 5 M NaCl and 250 µL Isopropanol were added prior to purification using solid phase extraction (SPE). These extractions are termed either RNASwift+Gu-HCl or RNASwift+Gu-HCl+DMSO for clarity. For the SPE, the sample mix was applied to a silica-membrane column (Qiagen/Invitrogen) and centrifuged at 13000 rpm for 1 minute. The flow through was discarded and 700 µL wash buffer, (15 mM TRIS-HCl, 85% ethanol, pH 7.4) added followed by centrifugation at 13000 rpm for 1 minute. The flow through was discarded and the dry column was re-centrifuged. The RNA was eluted with 100 µL RNase-free water (Ambion).