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Protocol for Synthesis of RNA
(with Sp6/T3/T7 RNA Polymerases)

1. Linearize template DNA with a restriction enzyme. Extract DNA with phenol/chloroform, and 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	10 µl
   4 NTP Mix, 10 mM each	10 µl (2 mM final concentration)
   linearized template DNA	1 µg
   RiboLock™ RNase Inhibitor	1.25 µl (50 u)
   SP6/T3/T7 RNA Polymerase	1.5 µl (30 u)
   DEPC-treated Water	to 50 µl

3. Incubate at 37°C for 2 hours.
4. Stop the reaction by the addition of 2 µl 0.5 M EDTA (pH 8.0) or by cooling at -20°C.
5. Analyze transcripts by electrophoresis.

Note

• The transcription reaction should be performed under conditions that exclude contamination with RNases. The tips, tubes and water should be nuclease-free. All the solutions should be prepared in DEPC-treated water. Wearing gloves is advisable.

• The reaction mixture should be prepared at room temperature, because DNA may precipitate in the presence of spermidine at 4°C.
• Under the conditions described above, more than 10 µg RNA per 1 µg template DNA is generated.
• The yield of the full-length transcripts decreases if template DNA is incompletely linearized due to a read-through reaction and accumulation of longer transcripts of variable length.
• The reaction mixture can be scaled up or down.

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 15:09