Total Bacterial RNA Labeling with Random Hexamers.

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Total Bacterial RNA Labeling with Random Hexamers.Arkady Khodursky, Ph.D.Email: khodursk@cmgm.stanford.eduUpdated: 2/10/9920-25ug of total RNA in 10-14 ul of diH20 should be purified using  RNA kitor hot phenol extraction. After obtaining RNA sample it is important to getrid of residual DNA with RNase-free DNAse followed by 1x PHOH,1xPHOH/CHCl3, 2x CHCl3 extraction. Extent of RNA purity achieved byPHOH-CHCl3 extraction is absolutely critical for successful labeling.Above indicated amount of RNA should be mixed with 500ng of random pdN6primer on ice in total volume of 15 ul.Incubate at 65 C for 10'.Incubate on ice for 2'.Add 3 ul of  1 mM FluoroLink Cy3 (orCy5) dUTP (Amersham Cat# PA53022).Add 11.6 ul an mix everything by pipeting 3-4 times on ice of reversetranscription mix:0.1 DTT - 3ul5x 1st strand buffer - 6 uldNTPs (25 mM dATP, 25 mM dCTP, 25 mM dGTP, 10 mMdTTP) - 0.6 ulSuperscriptII - 2 ul(All from GibcoBRL Cat.# 18064-014)Incubate the complete mixture for 10' at RT.Transfer to 42 C for 110'.Stop by adding 1.5 ul of 1n NaOH for 10' at 65 C.Neutralize with 1.5 ul of 1M HCL.

 

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