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Protocol for Synthesis of High Specific Activity Radiolabeled RNA
(with SP6/T3/T7 RNA Polymerases)

1. Linearize template DNA with a restriction enzyme. Extract DNA with phenol/chloroform, then with chloroform/isoamyl alcohol, and precipitate with ethanol. Dissolve DNA in DEPC-treated water.
2. Prepare the following reaction mixture:
   

   5X transcription buffer for RNA Polymerase	4 µl
   3 NTP Mix, 10 mM each (without labeled NTP)	1 µl (0.5 mM final concentration)
   100 µM CTP	2.4 µl (12 µM final concentration)
   [alfa-32P]-CTP, ~30 TBq/mmol (800 Ci/mmol)	1.85 MBq (50 µCi)
   linearized template DNA	0.2-1.0 µg
   RiboLock™ RNase Inhibitor	0.5 µl (20 u)
   SP6/T3/T7 RNA Polymerase	1 µl (20 u)
   DEPC-treated Water	to 20 µl

3. Incubate at 37°C for 2 hours.
4. Stop the reaction by cooling at -20°C.
5. Determine the percentage of the label incorporated into RNA.

Note

• RNA synthesized under the conditions described above usually has a specific activity of 3-5x10⁸ dpm/µg.
• RNA may be radiolabeled with [³²P], [³⁵S] or [³H]-ribonucleotides. The use of 1.85 MBq (50 µCi) of 5'-[alfa-³²P]-CTP, ~30 TBq/mmol (800 Ci/mmol), 11.1 MBq (300 µCi) of 5'-[alfa-³⁵S]-UTP, >37 TBq/mmol (>1000 Ci/mmol), 0.925 MBq (25 µCi) of 5,6-[³H]-UTP, 1.1-2.2 TBq/mmol (30-60 Ci/mmol) for 20 µl reaction mixture is recommended.
• The yield of the full-length transcripts is reduced when the concentration of labeled NTP is below 12 µM.

Reference

1. Melton, D.A., et al., Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter, Nucleic Acids Res.,12, 7035-7056, 1984.
2. Church, G.M., Gilbert, W., Genomic sequencing, Proc. Natl. Acad. Sci. USA, 81, 1991-1995, 1984.
3. Peebles, C.L., et al., A self-splicing RNA excises an intron lariat, Cell, 44, 213-223, 1986.
4. Melton, D.A., Injected antisense RNAs specifically block messenger RNA translation in vivo, Proc. Natl. Acad. Sci. USA, 82, 144-148, 1985.
5. Krieg, P.A., Melton, D.A., Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs, Nucleic Acids Res., 12, 7057-7070, 1984.
6. Krainer, A.R., et al., Normal and mutant human beta-globin pre-mRNAs are faithfully and efficiently spliced in vitro, Cell, 36, 993-1005, 1984.
7. Witherell, G.W., et al., Cooperative binding of R17 coat protein to RNA, Biochemistry, 29, 11051-11057, 1990.
8. Bernstein, E., et al., Role for bidentate ribonuclease in the initiation step of RNA interference, Nature, 409, 363-366, 2001.

Ordering Information

SP6 RNA Polymerase T3 RNA Polymerase T7 RNA Polymerase RiboLock™ RNase Inhibitor DNase I, RNase-free T7 Transciption Kit RiboRuler™ RNA Ladders NTP Set, molecular biology grade Agarase Pyrophosphatase, Inorganic (from yeast) 0.5 M EDTA, pH 8.0 DEPC-treated Water, molecular biology grade

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Updated sausio 31, 2008 15:15