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In vitro transcription of radiolabelled RNAs

Posted on Tuesday, October 31, 2006

Description
To produce radiolabelled RNA to be used as hybridization probes or substrates for binding reactions.

Procedure
Mix together in an RNase-free tube:

-DNA (roughly 50ng of PCR fragment or 1 ug plasmid)
-4 ul 5X transcription buffer
-1 ul NTP/DTT mix (see below)
- alpha-32P-UTP for a final volume of 20ul / reaction
-1 ul T7 RNAP (~25units/ul)

Incubate at 37 degrees C for 1 hr.

Assess % incorporation by HCl precipitation.



Recipes
NTP/DTT mix (remember that the stocks must be RNAse-free)
-1ul each 100mM NTP (so, a total of 4ul)
-1ul 1M DTT (dithiothreitol)
-5ul RNase-free water

10 mM NTPs are not especially stable. So use as much of this as you need for the transcriptions, then throw out the rest.

-5X buffer
-0.2 M Tris-HCl (pH 7.5)
-50mM NaCl
-30 mM MgCl2
-10 mM spermidine


Supplies
RNase-free tips and tubes.

Tips
Note that all solutions must be RNase-free. Please see protocol for avoiding RNase contamination.

If the RNA is to be used as a probe, it can be used straight from the reaction mixture.

If it is to be used in a binding reaction, it would be best to gel-purify to get rid of prematurely terminated RNAs and unincorporated nucleotides.

Submitted by: hollings

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