P-CAGE-25009 - P-CAGE-25009

1.Total RNA extraction

Grind 3-6g (about 12 plants) of Arabidopsis leaves in liquid N2 with mortar and pestle. Transfer to 50ml Falcon tube.

Add 18ml of 1:2:1 solution (precipitation at RT)

o 1 volume of 2M Tris-HCl pH 8.2

o 2 volumes of 0.5M EDTA pH 8.0 (DEPC treated)

o 1 volume of 20% SDS

Add 9 ml of phenol saturated with 0.1M Tris-HCl pH 8.2. Stir for minimum 15min.

Add 9ml chloroform. Stir for minimum 10min.

Centrifuge 3600 x g 15min.

Treat upper phase (aqueous) twice with 9ml chloroform, centrifuging 3600 x g 15min.

Transfer 15ml of aqueous phase to 30ml disposable polypropylene tube and add equal volume of ice-cold 6M LiCl (DEPC treated). Precipitate O/N on ice at 4°C.

Centrifuge 9800 x g 20min 4°C.

Resuspend into 2.5ml H2O, add 0.25ml 3M NaAcetate pH 5.2 and 3 volumes (8.3ml) of EtOH. Precipitate at RT for ca 60min.

Centrifuge 9800 x g 20min 16°C, rinse with 5ml 70% EtOH, centrifuge 9800 x g 5min, air dry pellet 15min and resuspend in 0.5 ml H2O.

Quantify RNA using Nanodrop. Dilute 2µl in 400µl 10 mM Tris pH 8.

2.Total RNA purification-Use Rneasy Mini Kit (QIAGEN)

mix thoroughly 150 µg total RNA with 350 µl Buffer RLT/1% beta-Mercaptoethanol

Add 250 µl ethanol 100% to each sample, mix by pipetting

Apply to an Rneasy mini column, centrifuge 8000 x g 15s, discard the flowthrough

Wash the column with 500 µl Buffer RPE, centrifuge 8000 x g 15s, discard the flowthrough

Add another 500 µl RPE buffer, centrifuge 8000 x g 2 min to dry the silica-gel membrane

Transfer the column into a new 2 ml collection tube and centrifuge 8000 x g 1 min to eliminate any Buffer RPE carryover

Elution : transfer the column into a 1.5 ml collection tube, add 40 µl H2O nuclease free 50°C preheated, centrifuge 8000 x g 1 min (twice)

Measure concentration (0.5 µl in 200 µl 10 mM Tris pH8)

Amplification, Extracted product
All experiments using protocol P-CAGE-25009: (E-ATMX-36, E-ATMX-37, E-ATMX-9)