E.coli Total RNA Labeling Protocol for Spotted Microarray

• If RNA is in ethanol, spin down 20 mg of RNA per reaction @ 14000 rpm for 20 min. at 4^o C.
• Pipette off supernatant and wash pellet with 100ml of 70% ETOH. (prepared with DEPC H₂ O)
• Spin 5 min. and remove supernatant without disturbing pellet
• Air dry pellet 15-20 min at Room Temp (RT). (Caution: if pellet is over dried it is hard to resuspend!)
• Resuspend RNA pellet in 12.5 ml DEPC H₂ O

RNA		12.5ul
Random Hexamer	5mg/ml	1ul
Labeling Control (Yeast RNA mix)		1ul
Total		17ul

Heat to RNA to 70 ^o C 5 min, ice 2 min, pulse spin

1X labeling mix

First Strand Buffer	5x	8ul
DTT	0.1M	4ul
dNTPs(low dTTP~undefined~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0~Kdiv~M~2~1~0~0~0~0~0~0~0~0~0~0~010x~K~Hdiv~M~2~1~0~0~0~0~0~0~0~0~0~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0~Kdiv~M~2~1~0~0~0~0~0~0~0~0~0~0~04ul~K~Hdiv~M~2~1~0~0~0~0~0~0~0~0~0~K~Htd~M~2~1~0~0~0~0~0~0~0~0~K~Htr~M~2~1~0~0~0~0~0~0~0~0~Ktr~M~2~1~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0RNAsin~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0	1ul
Total		17ul

• To the RNA/hexamer mix add 17.0 ml of labeling mix and incubate 10min at RT
• Add 1.5 ml of appropriate CyDye dUTP (1mM stock) followed by 1.5 ml SSII reverse transcriptase, mix well by tapping and pulse spin
• Incubate 1hr at 42 ^o C in the dark
• Add an additional 1.5 ml SSII reverse transcriptase, tap, pulse spin and continue incubation 1hr
• Degrade RNA by addition of 2 ml 1N NaOH, vortex, pulse spin and incubate 15 min at 65 ^o C
• Neutralize by addition of 2 ml 1N HCl, vortex and pulse spin

• Prewash Microcon-30 microfilter by adding 450ml miliQ H₂ O and spinning for 10 min. @ 12,000 RPM.
• Add 450ml miliQ H₂ O 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 H₂ O 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.
• Spin 10 minutes at 12,000 RPM
• Repeat step 4 , spin 12min to get smaller volume.
• Invert column into a fresh tube and spin 1 minute at maximum speed to recover probe. Carefully measure recovered probe volume if necessary.
• Transfer recovered probe to the appropriate partner probe.

Note: Probes can be combined after the first wash step.

Probe can be stored at 4C or -20C in dark for further purpose.

 

Cy3-dUTP	1mM	Perkinelmer	NEL578
Cy5-dUTP	1mM	Perkinelmer	NEL579
SuperScript II	200U/ul	Invitrogen	18064-014
RNAsin	20-40U/ul	Promega	N2515
100 mM dNTP seundefined~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~010X~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0Amersham~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~027~F2035~F01~K~Htd~M~2~1~0~0~0~0~0~0~0~0~K~Htr~M~2~1~0~0~0~0~0~0~0~0~Ktr~M~2~1~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0pd~AN~B~Ksub~M6_~K~Hsub~M_Sodium_Salt_~AHexamer~B~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~050U~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0Amersham~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~027~F2166~F01~K~Htd~M~2~1~0~0~0~0~0~0~0~0~K~Htr~M~2~1~0~0~0~0~0~0~0~0~Ktr~M~2~1~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0Microcon_YM~F30_column~K~Htd~M~2~1~0~0~0~0~0~0~0~0~0~Ktd~M~2~1~0~0~0~0~0~0~0~0~0~0	Amicon	42410

 

Other reagents: 20X SSC, TE pH7.4, 10% SDS, 500 mM EDTA, 1M NaOH, 1M Tris-HCl pH7.5, sterile dH2O and DEPC H2O

~undefined comes lyophilized, must be resuspended at specified concentration.

~undefinedfor_10X_stock~I_5_mM_each_of_dA~E_dG~E_dC_and_2_mM_of_dT_in_DEPC_H2O~K~Hp~M~2~1~0~0~K~Hdiv~M~2~1~0~K~Hdiv~M~2~1~K~Hdiv~M~2~1~Kp~M~2~1~0 

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