Isolation of total RNA from Synechocystis sp. PCC 6803 Can be used for further work, like qPCR Always work fast, with ice and RNase-free (wear gloves!)PGTX solution (100 mL) 39.6 g phenol, 6.9 mL glycerol, 0.1 g hydroxyquinoline, 0.58 g EDTA, 0.8 g NaOAc, 9.5 g guanidine thiocyanate, 4.6 g guanidine hydrochloride PGTX contains phenol, wear safety gear and glovesSynechocystis cultureChloroform\/IAAIPA + NaOAc (in a specific amount) 1 vol. IPA or 3 vol. EtOH and NaOAc 30:1 or 10:1 4.6 g guanidine hydrochloride PGTX contains phenol, wear safety gear and glovesSynechocystis cultureChloroform\/IAAIPA + NaOAc (in a specific amount) 1 vol. IPA or 3 vol. EtOH and NaOAc 30:1 or 10:1Synechocystis cultureChloroform\/IAAIPA + NaOAc (in a specific amount) 1 vol. IPA or 3 vol. EtOH and NaOAc 30:1 or 10:1StartCentrifuge 5 ml of culture (oD750 of~ 1) for 5 minutes at maximum speed (T = 4\u00b0C)Discard supernatant. Resuspend cyanobacterial pellet in the remaining water (~1 ml). Transfer to a fresh 2 ml tube (RNase-free). Spin down 1 min at maximum speed.Discard remaining supernatant. resuspend pellet in 1 ml PGTX solution. Flash freeze and store at -80\u00b0C for later extraction, or proceed with the next step.ExtractHeat samples at 95\u00b0C for 5 minutes in a shaking heat block. Vortex samples from time to time to ensure complete lysis.Place samples on ice for 5 minutes.Add 700 \u00b5l Chloroform\/IAA. Vortex sample until it is opaque. Incubate at RT for 10 min, vortexing from time to time.Centrifuge samples at maximum speed for 10 minutes to seperate phases. Transfer aqueous phase (600 \u00b5l) to a fresh tube (RNase-free).Add 1 vol. (600\u00b5l) Chloroform\/IAA. Mix well by vortexing. Centrifuge 10 minutes at maximum speed. Transfer aqueous phase (500\u00b5l) to a fresh tube (RNase-free).PrecipitationAdd 517 \u00b5l IPA + NaOAc to the samples. Mix well. Precipate over weekend at -20\u00b0C. Also possible: 1h at -80\u00b0C or over night at -20\u00b0C.Centrifuge precipitated sample at 4\u00b0C and maximum speed for at least 30 min. Remove supernatant, making sure not to disrupt the RNA pellet.Wash pellet with 70% EtOH (300\u00b5l). Centrifuge for 15 minutes, 4\u00b0C at maximum speed. Completely remove supernatant. Dry at RT for ~5 minutes. Do not overdry! Resuspend pellet in 40 \u00b5l pure, RNase-free water. Store at -80\u00b0C.