E.coli Total RNA Labeling Protocol for Spotted Microarray
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1025
Note:
Start with 20 mg of total RNA for each labeling reaction.
All solutions that can be filtered should be filtered.
Cy dyes are light sensitive and should ALWAYS be handled in dim light.
RNA Preparation
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If RNA is in ethanol, spin down 20 mg of RNA per reaction @ 14000 rpm for 20 min. at 4o C.
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Pipette off supernatant and wash pellet with 100ml of 70% ETOH. (prepared with DEPC H2 O)
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Spin 5 min. and remove supernatant without disturbing pellet
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Air dry pellet 15-20 min at Room Temp (RT). (Caution: if pellet is over dried it is hard to resuspend!)
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Resuspend RNA pellet in 12.5 ml DEPC H2 O
RNA Random Hexamer Labeling Control (Yeast RNA mix)
Total Heat to RNA to 70 o C 5 min, ice 2 min, pulse spin
Labeling
Prepare labeling mix (prepare 1 labeling mix for all smaples labeled at the same time).
1X labeling mix
First Strand Buffer DTT 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
Total
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To the RNA/hexamer mix add 17.0 ml of labeling mix and incubate 10min at RT
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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
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Incubate 1hr at 42 o C in the dark
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Add an additional 1.5 ml SSII reverse transcriptase, tap, pulse spin and continue incubation 1hr
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Degrade RNA by addition of 2 ml 1N NaOH, vortex, pulse spin and incubate 15 min at 65 o C
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Neutralize by addition of 2 ml 1N HCl, vortex and pulse spin
Clean up Labeled Probes
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Prewash Microcon-30 microfilter by adding 450ml miliQ H2 O and spinning for 10 min. @ 12,000 RPM.
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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)
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Spin at 12,000 RPM in microfuge for 10 minutes or until 20-40 ml remains in the filter.
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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.
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Spin 10 minutes at 12,000 RPM
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Repeat step 4 , spin 12min to get smaller volume.
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Invert column into a fresh tube and spin 1 minute at maximum speed to recover probe. Carefully measure recovered probe volume if necessary.
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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.
Reagents and Suppliers
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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