E.coli Total RNA Labeling Protocol for Spotted Microarray- 点击： 作者：51protocol收集 来源： 日期：2007-03-13 本站论坛
E.coli Total RNA Labeling Protocol for Spotted Microarray
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.
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If RNA is in ethanol, spin down 20 mg of RNA per reaction @ 14000 rpm for 20 min. at 4oC.
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Pipette off supernatant and wash pellet with 100ml of 70% ETOH. (prepared with DEPC H2O)
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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 H2O
RNA
12.5ul
Random Hexamer
5mg/ml
1ul
Labeling Control (Yeast RNA mix)
1ul
Total
17ul
Heat to RNA to 70 oC 5 min, ice 2 min, pulse spin
Prepare labeling mix (prepare 1 labeling mix for all smaples labeled at the same time).
First Strand Buffer
5x
8ul
DTT
0.1M
4ul
dNTPs(low dTTP)**
10x
4ul
RNAsin
1ul
Total
17ul
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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 oC 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 oC
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Neutralize by addition of 2 ml 1N HCl, vortex and pulse spin
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