Qiagen Omniscript Reverse Transcription: FGB 03/25/02

Reagent: 2X 4X f.c. Master Mix:

10X buffer 4 8 X

dNTPs 4 8 .5mM

oligo-dT (20X P.E.=50uM)* 1 2 uM

Rnase Inhibitor (P.E.=20U/ul)** 1 2 0U/2Xrxn

RT enzyme 2 4 U/2Xrxn

RNA (diluted to 0.1ug/ul) (20ul)*** (20) 25ng/ul

DH2O 8 36 to 40 ul

Final volume= 40ul 80ul

* oligo-dT may come as 10X or 20X so dilute accordingly.

** Some are 10U/ul. If more concentrated than 20U/ul, dilute Rnase inhibitor in 1X RT.

***For a 2X reaction, DNase 2ug in 20ul for f.c. of 0.1ug/ul (2X=40ul).

Incubate at 37’C for 1 hour, 93’C for 5 minutes then immediately on ice.

Note: Dilute final RT reaction 5X to 10ng/ul and use 10ul (100ng).