Total Bacterial RNA Labeling with Random Hexamers.

Arkady Khodursky, Ph.D.
Email: khodursk@cmgm.stanford.edu

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.