E.coliTotalRNALabelingProtocolforSpottedMicroarrayIf RNA is in ethanol, spin down 20 mg of RNA per reaction @ 14000 rpm for 20 min. at 4oC.Pipette off supernatant and wash pellet with 100ml of 70% ETOH. (prepared with DEPC H2O)Air dry pellet 15-20 min at Room Temp (RT). (Caution: if pellet is over dried it is hard to resuspend!)Prepare labeling mix (prepare 1 labeling mix for all smaples labeled at the same time). Add 1.5 ml of appropriate CyDye dUTP (1mM stock) followed by 1.5 ml SSII reverse transcriptase, mix well by tapping and pulse spinAdd an additional 1.5 ml SSII reverse transcriptase, tap, pulse spin and continue incubation 1hrDegrade RNA by addition of 2 ml 1N NaOH, vortex, pulse spin and incubate 15 min at 65 oCPrewash Microcon-30 microfilter by adding 450ml miliQ H2O and spinning for 10 min. @ 12,000 RPM.Add 450ml miliQ H2O to each of the probe samples (or total 500ul). Mix thoroughly by pipetting up and down. Transfer samples to separate Microcon-30 microfilters. (Amicon)Spin at 12,000 RPM in microfuge for 10 minutes or until 20-40 ml remains in the filter.Add 450 ml miliQ H2O to the probe and gently mix by pipetting up and down. Be careful not to touch the filter at the bottom of the filtration unit.Invert column into a fresh tube and spin 1 minute at maximum speed to recover probe. Carefully measure recovered probe volume if necessary.Other reagents: 20X SSC, TE pH7.4, 10% SDS, 500 mM EDTA, 1M NaOH, 1M Tris-HCl pH7.5, sterile dH2O and DEPC H2O* comes lyophilized, must be resuspended at specified concentration.