Question

In vitro transcription:

.If you wanted to ensure that you only synthesized “insert RNA”, what steps would have to be taken?

Answer 1

The steps that should be taken in synthesis are

Digest the (RNase-free) plasmid DNA (e.g. 100 μg) with
an appropriate restriction enzyme that cleaves downstream
of the T7 promoter and the segment to be transcribed.
2. Add proteinase K to a final concentration of 50 μg/mL and
incubate for 30 min at 37◦C in order to remove the restric-
tion enzyme from the template DNA.
3. Extract twice with one volume of phenol-chloroform (see
Note 6).
4. Precipitate the template with 2.5 vols of 96% ethanol.
5. Resuspend the DNA to 1 μg/μL in TE 8.0.
6. Run an aliquot (e.g. 0.5 μg) of the DNA on an agarose gel
to check the linearization of the plasmid
PCR Templates for
In Vitro Transcription
1. Design the oligos for PCR-amplification.
2. Make a standard PCR reaction.
3. Purify the PCR product using a commercial PCR clean-up
kit (GenEluteTM PCR Clean-Up Kit Sigma) according to
the manufacturer’s instruction

Set up the transcription reaction by adding the components in a siliconized or Teflon-coated tube in the following order at room temperature

5 μL of 5× transcription buffer
– 4 μL of 10× rNTP mix
– 2.5 μL of 100 mM DTT
– 11.5 μL DEPC-treated dH2O
– 1 μL of template DNA (linearized plasmid or PCR-product)
– 1 μL 10 U of the appropriate (in this case T7) RNA poly-
merase (see Note 9)
– Incubate for 30–60 min at 37◦C.
2. In Vitro
Transcription of
32P-Labelled Transcripts
1. Set up the transcription reaction by adding at room tempera-
ture the components in a siliconized or Teflon-coated tube in the
following order:
– 5 μL of 5× transcription buffer
– 4 μL of 10× rNTP mix “low UTP”
– 2.5 μL of 100 mM DTT
– 6.5 μL DEPC-treated dH2O
– 1 μL of template DNA (linearized plasmid or PCR-product)
– 5 μL of 3,000 Ci/mmol, 10 mCi/ml [α-
32P]UTP
– 1 μL 10 U of the appropriate RNA
polymerase
2. Incubate for 30–60 min at 37◦C.