MiniV™ In Vitro Transcription Kit

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Last modified: 2008-07-15 14:57:15

MiniV™ In Vitro Transcription Kit provides MiniV™ RNA Polymerase* (MiniV RNAP) and all other components for in vitro transcription of RNA using single-stranded DNA (ssDNA) templates that are functionally linked to a single-stranded bacteriophage N4 promoter. MiniV™ RNAP is a transcriptionally-active 1,106-amino acid domain of the N4 virion RNA polymerase. Like the N4 virion enzyme from which it is derived, MiniV™ RNAP retains the unique capability to transcribe ssDNA templates having a single-stranded N4 promoter.

Since it lacks RNA strand-displacement or unwinding activity on RNA:DNA hybrids, MiniV™ RNAP requires E. coli Single-Stranded Binding Protein (EcoSSB Protein) to displace the RNA transcript from the DNA template strand for efficient in vitro transcription. The amount of EcoSSB-activated displacement appears to vary with the total length of the template strand and the length of the transcript. RNA synthesis by MiniV™ RNAP also requires ribonucleoside triphosphates and divalent Mg+2.

Linear templates of any length that contain an appropriate N4 promoter can be transcribed. Though RNA yield from a MiniV™ in vitro transcription reaction varies depending on the sequence of the template, typically 1-5 moles of RNA are produced per mole of linear ssDNA template. Like other RNA polymerases, MiniV™ RNAP can also synthesize concatameric RNA by rolling circle transcription of circular single-stranded DNA templates (such as circular templates produced using CircLigase™ ssDNA Ligase).

Figure 1. MiniV™ transcription of a single-stranded DNA oligo template. A 71-nt single-stranded oligodeoxynucleotide template containing the phage N4 P2 promoter sequence was transcribed for 1 hour at 37°C in a standard 20-µl reaction containing 100 pmole MiniV™ RNA Polymerase, 0.75 mM of each NTP and 20 ng/µl EcoSSB Protein. Reaction products were visualized on a 20% acrylamide-8 M urea denaturing gel stained with SYBR® Gold. Lane 1: 71-base single-stranded oligo template (control template provided in the MiniV N4 Transcription Kit). Lane 2: The expected 43-base RNA transcript was produced.

Last modified: 2008-05-14 16:46:46

Protocols for: MiniVr™ In Vitro Transcription Kit

(catalogue number MV41025)

Please note: all protocols off site are the responsibility of the products supplier

Last modified: 2008-05-14 16:46:46

References

1. Kazmierczak, K.M., et al. (2002) EMBO J., 21, 5815.
2. Davidova, E. and Rothman-Denes, (2003) Proc. Natl. Acad. Sci. USA, 100, 9250.
3. Haynes, et al. (1985) Cell 41, 597.
4. Glucksmann, M.A. et al. (1992) Cell 70, 491.

Last modified: 2008-05-14 16:46:46