TITLE: Extraction of total RNA from E. coli cells
AUTHORS: Alice Pawlowski

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The protocol is used for the extraction of total RNA from </span><span style = "font-style:italic;">E. coli</span><span> cells. It is based on the method described by Chomczynski and Sacchi, 1987 (</span><a href="https://doi.org/10.1016/0003-2697(87)90021-2" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">https://doi.org/10.1016/0003-2697(87)90021-2</span></a><span style = ":UNDERLINE;">). </span><span>Total RNA is isolated from cells expressing small regulatory RNAs (trRNAs) either constitutively (E. coli W3110) or after induction with anhydrotetracycline (E. coli W1310 Z1). </span></div></div>

[STEPS]
?. [stop of RNA synthesis]
work under the hoodmix 1 ml of cells with 200 µl 'stopmix'- solution (5 % phenol in ethanol) in a 2 ml safe lock tube tube → stops RNA production in the cellscentrifuge for 5 min at 4°C and 14000 x gdiscard the supernatant and resuspend the pellet in 1 ml NucleoZOL (Macherey and Nagel),  place on ice (better for rapid freezing: dry ice or liquid nitrogen)→ store cells at -20 or  -80 °C (only for a short time, maximum 2 weeks)  or proceed to next step
?. [RNA-isoloation]
incubate the sample at 65 °C and 250 rpm (Thermomixer) for 10 minmix with 400 µl Phenol-Chloroform/Isoamylalcohol (Roti®-)Aqua-P/C/I) by inverting for 10 scentrifuge at 4°C for 10 min at 14000 x gtransfer aqueous (upper) phase to a fresh 1,5 ml safe lock reaction tube, work on icemix with 450 µl Chloroform/Isoamylalcohol (Roti®-C/I)centrifuge at 4°C for 10 min at 14000 x gtransfer aqueous (upper) phase to a new reaction tube and add 1 Vol. icecold Isopropanol + 1/10 vol (e.g. 20 µl for 200 µl Isopropanol) 3 M Na-Acetat (pH 5.2), mix and store at least 30 min at  -20 °C or -80 °Ccentrifuge at 4°C for 30 min at 14000 x gremove the supernatant (take care of the RNA-pellet) and add 350 µl of icecold 75% Ethanolcentrifuge for 5 min at 4°C and 15000 rpmadd again 350 µl of icecold 75% etahnol and centrifuge for 5 min at 4°C and 15000 rpmremove the supernatant and dry the pellet at room temperature for ca.15 minresuspend  the pellet in 30 µl Molecular Biology Grade Water and store at -80°C
?. 
?. [incubation of cells ]
freshly transform E. coli cells with plasmids encoding trRNA sequences or empty vectorinoculate 3 ml LB-vegetal medium + antibiotic (25 µg/ml Km for W1310, 25 µg/ml + 100 µg/ml Spec for W1310Z1) in culture tubes with a single colonyincubate o/n at 37°C and 230 rpmfor constitutive RNA-synthesis (E. coli W3110): dilute 1:100 in fresh medium with 25 µg/ml Km and incubate until OD600 reaches 0.7 - 1.0 (in culture tubes or 6 well plates), proceed to step 2for induced RNA-synthesis (E. coli W3110Z1): dilute 1:50 in fresh medium ( 200 µl o/n culture in 10 ml medium + antibiotic in 100 ml Erlenmeyer flask) and incubate until OD600 reaches 0.4 - 0.6if you have several different cultures that do not grow at same speed: store samples on ice until the last one reaches the defined OD (keep that one on ice as well for 10 min)meanwhile prepare 12 well plates with inducer: pipet in each well of a row 10 µl of either         1) EthOH,         2) aTC (= 200 ng/ml final concentration)        3) IPTG ( = 100 µM final concentration)        4) aTc + IPTG ( 200 ng/ml, 100 µM final concentration)add 1 ml of culture and incubate at 37°C and 230 rpm for 1 hproceed with step 2for constitutive RNA-synthesis (E. coli W3110): dilute 1:100 in fresh medium with 25 µg/ml Km and incubate until OD600 reaches 0.7 - 1.0 (in culture tubes or 6 well plates), proceed to step 2for induced RNA-synthesis (E. coli W3110Z1): dilute 1:50 in fresh medium ( 200 µl o/n culture in 10 ml medium + antibiotic in 100 ml Erlenmeyer flask) and incubate until OD600 reaches 0.4 - 0.6if you have several different cultures that do not grow at same speed: store samples on ice until the last one reaches the defined OD (keep that one on ice as well for 10 min)meanwhile prepare 12 well plates with inducer: pipet in each well of a row 10 µl of either         1) EthOH,         2) aTC (= 200 ng/ml final concentration)        3) IPTG ( = 100 µM final concentration)        4) aTc + IPTG ( 200 ng/ml, 100 µM final concentration)add 1 ml of culture and incubate at 37°C and 230 rpm for 1 hproceed with step 2for induced RNA-synthesis (E. coli W3110Z1): dilute 1:50 in fresh medium ( 200 µl o/n culture in 10 ml medium + antibiotic in 100 ml Erlenmeyer flask) and incubate until OD600 reaches 0.4 - 0.6if you have several different cultures that do not grow at same speed: store samples on ice until the last one reaches the defined OD (keep that one on ice as well for 10 min)meanwhile prepare 12 well plates with inducer: pipet in each well of a row 10 µl of either         1) EthOH,         2) aTC (= 200 ng/ml final concentration)        3) IPTG ( = 100 µM final concentration)        4) aTc + IPTG ( 200 ng/ml, 100 µM final concentration)add 1 ml of culture and incubate at 37°C and 230 rpm for 1 hproceed with step 2        1) EthOH,         2) aTC (= 200 ng/ml final concentration)        3) IPTG ( = 100 µM final concentration)        4) aTc + IPTG ( 200 ng/ml, 100 µM final concentration)add 1 ml of culture and incubate at 37°C and 230 rpm for 1 hproceed with step 2