TITLE: RNA extraction with PGTX
AUTHORS: Lutz Berwanger

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used for RNA extraction in cyanobacteria after Pinto et al. 2009.</div><div class = "text-block"><span style = "font-weight:bold;">Pinto, Fernando Lopes; Thapper, Anders; Sontheim, Wolfgang; Lindblad, Peter (2009): Analysis of current and alternative phenol based RNA extraction methodologies for cyanobacteria. In: </span><span style = "font-weight:bold;font-style:italic;">BMC molecular biology </span><span style = "font-weight:bold;">10, S. 79. DOI: 10.1186/1471-2199-10-79</span><span style = "font-weight:bold;"> </span></div><div style = "text-align :; float : ;"><img style = "" src = "data:image/png;base64,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" /></div><div class = "text-block">.</div></div>

[STEPS]
?. Centrifuge for 3 minutes at 4˚C and 4.000 g.
?. Resuspend the cell pellet in 1 ml of PGTX. Freeze in liquid nitrogen and store at -20 ˚C.
Wear goggles, a lab coat and gloves when dealing with PGTX and liquid nitrogen.
?. Incubate for 5 min at 95 °C, shaking at 250 rpm in Thermomixer (Eppendorf)
?. Rapidly chill 5 min on ice.
?. Add 700 µl of Roti-C/I
Wear goggles, a lab coat and gloves when dealing with phenol and chloroform.
?. Let the samples incubate for 10 min at room temperature in a Thermomixer. Vortex from time to time.
?. Centrifuge for 15 min at 14.000 g, 4 ˚C. Transfer the upper aqueous phase (contains RNA) to a fresh reaction tube and add the same volume (450 µL) of Aqua- C/I (Chloroform/Isoamylalcohol).
Wear goggles, a lab coat and gloves when dealing with phenol and chloroform.
?. Thoroughly mix by vortexing. Centrifuge for 15 min at high speed. Transfer the upper aqueous phase to a 1.5 mL reaction tube.
Wear goggles, a lab coat and gloves when dealing with phenol and chloroform.
?. Add 1 volume of isopropanol.
?. Mix and incubate for at least 30 min at -20 ˚C. (Can be left overnight.)
?. Centrifuge at 14.000 g for 30 min.
?. Wash with 75% chilled ethanol. Avoid resuspending the pellet.
?. Discard supernatant.
?. Centrifuge at 14.000 g.
?. Repeat washing step with 75% chilled ethanol.
Remove excess of EtOH by using a pipet.
?. Air dry pellet at RT. Do not overdry!
?. Resuspend the pellet with 30 µL volume of ddH2O.
Usually 40 µL DECP-treated H2O
?. Fill 50 mL Falcon tube with ice. Fill with bacterial liquid culture, up to a volume of ~45 mL.
?. Discard supernatant. Resuspend cell pellet in residual water (~1 mL). Transfer to 2 mL 'safe lock' tube.
?.