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Total Bacterial RNA Labeling with Random Hexamers. Arkady Khodursky, Ph.D. Email: firstname.lastname@example.org Updated: 2/10/99 20-25ug of total RNA in 10-14 ul of diH20 should be purified using RNA kit or hot phenol extraction. After obtaining RNA sample it is important to get rid of residual DNA with RNase-free DNAse followed by 1x PHOH, 1xPHOH/CHCl3, 2x CHCl3 extraction. Extent of RNA purity achieved by PHOH-CHCl3 extraction is absolutely critical for successful labeling. Above indicated amount of RNA should be mixed with 500ng of random pdN6 primer 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 reverse transcription mix: 0.1 DTT - 3ul 5x 1st strand buffer - 6 ul dNTPs (25 mM dATP, 25 mM dCTP, 25 mM dGTP, 10 mMdTTP) - 0.6 ul SuperscriptII - 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.