2.0µl 5x Transcription buffer
1.0µl DTT, 100mM
1.0µl DNA (linearized plasmid 1µg/µl)
2.0µl GTP+CTP+ATP Mix (stock solution containing 2.5mM @)
2.0µl Sterile dH2O
Mix thoroughly and centrifuge.
Add 1.0µl RNA Polymerase (SP6, T7 or T3 as required)
Mix gently by pipeting, do not vortex, and incubate 1-2 hours, 37°C.
(Order all of the above as ready-made stocks from Promega Biotech).
Add 1.0µl RQ1 DNase to the transcription reaction above
Incubate 15 min., 37°C.
To extract RNA after DNase step, add to the reaction:
20µl 1x TE
1.0µl tRNA (50mg/ml)
Invert column gently for 20-25 times to suspend the gel.
Remove TOP cap first, followed by the bottom cap.
Allow the fluid within the column to drip through by gravity.
Cut a collection tube (comes with column) approx. 5mm from bottom, place column in the collection tube and place the assembly in a 15ml tube.
Spin for 2 min at 1100g or 2500rpm (setting #6 on IEC centrifuge).
Discard fluid and collection tube.
Take 1µl from reaction and place in microfuge tube.
Add 99µl 1xTE.
Pipet 1.0µl of 1/100 dilution onto a small piece of DE81 ion exchange paper
Wash 3 times 5 min. in 0.5M NaPO4, pH7.4
Wash 10 seconds in dH2O
Rinse briefly (<10sec) in 100% etoh
Dry throughly and count in 10ml scintillation fluid
Thaw the riboprobe and keep on ice.