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 37íC for 1 hour, 93íC for 5 minutes then immediately on ice.

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