TITLE: Isolation of total RNA from Synechocystis
AUTHORS: iGEM Dusseldorf

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-style:italic;">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!)</span></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">PGTX solution (100 mL)</li><li style = "counter-reset:ol0;list-style-type:disc;">    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,  </li><li style = "counter-reset:ol0;list-style-type:disc;">           4.6 g guanidine hydrochloride</li><li style = "counter-reset:ol0;list-style-type:disc;">    PGTX contains phenol, wear safety gear and gloves</li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;">Synechocystis culture</li><li style = "counter-reset:ol0;list-style-type:disc;">Chloroform/IAA</li><li style = "counter-reset:ol0;list-style-type:disc;">IPA + NaOAc (in a specific amount)</li><li style = "counter-reset:ol0;list-style-type:disc;">    1 vol. IPA or 3 vol. EtOH and NaOAc 30:1 or 10:1</li></ul></div><div class = "text-block">           4.6 g guanidine hydrochloride</div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">    PGTX contains phenol, wear safety gear and gloves</li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;">Synechocystis culture</li><li style = "counter-reset:ol0;list-style-type:disc;">Chloroform/IAA</li><li style = "counter-reset:ol0;list-style-type:disc;">IPA + NaOAc (in a specific amount)</li><li style = "counter-reset:ol0;list-style-type:disc;">    1 vol. IPA or 3 vol. EtOH and NaOAc 30:1 or 10:1</li></ul></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Synechocystis culture</li><li style = "counter-reset:ol0;list-style-type:disc;">Chloroform/IAA</li><li style = "counter-reset:ol0;list-style-type:disc;">IPA + NaOAc (in a specific amount)</li><li style = "counter-reset:ol0;list-style-type:disc;">    1 vol. IPA or 3 vol. EtOH and NaOAc 30:1 or 10:1</li></ul></div><div class = "text-block"><span style = "font-weight:bold;">Start</span></div><div class = "text-block"><span>Centrifuge 5 ml of culture (oD</span><span style = "vertical-align:sub;">750</span><span> of~ 1) for 5 minutes at maximum speed (T = 4°C)</span></div><div class = "text-block">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.</div><div class = "text-block">Discard remaining supernatant. resuspend pellet in 1 ml PGTX solution. Flash freeze and store at -80°C for later extraction, or proceed with the next step.</div><div class = "text-block"><span style = "font-weight:bold;">Extract</span></div><div class = "text-block">Heat samples at 95°C for 5 minutes in a shaking heat block. Vortex samples from time to time to ensure complete lysis.</div><div class = "text-block">Place samples on ice for 5 minutes.</div><div class = "text-block">Add 700 µl Chloroform/IAA. Vortex sample until it is opaque. Incubate at RT for 10 min, vortexing from time to time.</div><div class = "text-block">Centrifuge samples at maximum speed for 10 minutes to seperate phases. Transfer aqueous phase (600 µl) to a fresh tube (RNase-free).</div><div class = "text-block">Add 1 vol. (600µl) Chloroform/IAA. Mix well by vortexing. Centrifuge 10 minutes at maximum speed. Transfer aqueous phase (500µl) to a fresh tube (RNase-free).</div><div class = "text-block"><span style = "font-weight:bold;">Precipitation</span></div><div class = "text-block">Add 517 µl IPA + NaOAc to the samples. Mix well. Precipate over weekend at -20°C. </div><div class = "text-block">Also possible: 1h at -80°C or over night at -20°C.</div><div class = "text-block">Centrifuge precipitated sample at 4°C and maximum speed for at least 30 min. Remove supernatant, making sure not to disrupt the RNA pellet.</div><div class = "text-block">Wash pellet with 70% EtOH (300µl). Centrifuge for 15 minutes, 4°C at maximum speed. Completely remove supernatant. Dry at RT for ~5 minutes. Do not overdry! Resuspend pellet in 40 µl pure, RNase-free water. Store at -80°C.</div></div>

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