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500 pg virus measured as protein)

50 pi

• 1 mg/ml inorganic pyrophosphatase

1 Ml

6. Incubate the mixture at 45 °C for 2h with shaking every 1 5 min to resuspend cores.

7. Recover and store the capped, radiolabeled reovirus mRNAs as described in Protocol 1, steps 3-8 except in step 6 collect 0.5 ml fractions from the Sephadex column.

To obtain the [3H] methyl- and 32P-labelled m7GpppGm caps, the reovirus transcripts are digested with nuclease PI and the caps are isolated as described in Section 4.

2.5 Synthesis of capped mRNAs using vaccinia virus

In vitro transcription using purified vaccinia virus yields poly(A)+ mRNAs containing m7GpppAm and m7GpppGm in a 2:1 ratio (29). These two cap forms may be released from the transcripts by nuclease P1 digestion and are readily resolved by paper chromatography (see Section 4.3.2). The transcription procedure is described in Protocol 3.

Protocol 3. In vitro synthesis of capped vaccinia virus poly (A)+ mRNAs

Equipment and reagents

• Vaccinia virions (2-4 mg/ml calculate from the A260 value where 1 mg/ml is equivalent to —15.7 A260 units) purified by standard methods as described in ref. 26

• 1.0 M Tris-HCI, pH 8.0, 0.1 M MgCI2, 40 mM stock solutions of ATP, CTP, GTP, and UTP, [methyl-3H]AdoMet, phenol ¡chloroform, elution buffer, Sephadex G-100 column3 as described in Protocol 1

Protocol 3. Continued Method

Set up the transcription reaction by mixing the following reagents in a

microcentrifuge tube on ice:

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