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进口试剂

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MiniV™ In Vitro Transcription Kit

进口试剂

MiniV™ In Vitro Transcription Kit

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产品名称:

MiniV™ In Vitro Transcription Kit

货号
MV41025
规格
25 Rxns
存储温度
-20℃
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产品描述

Applications

  • Synthesis of RNA from ssDNA linked to a bacteriophage N4 promoter.

The MiniV™ In Vitro Transcription Kit provides MiniV™ RNA Polymerase* (MiniV RNAP) and all other components for in vitro transcription of RNA using 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.1 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 (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.2 RNA synthesis by MiniV RNAP also requires NTPs and Mg2+.

Linear templates of any length that contain an appropriate N4 promoter can be transcribed. Though RNA yield from a MiniV in vitrotranscription 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 ssDNA templates (such as circular templates produced using CircLigase™ ssDNA Ligase).

Phage N4 transcription promoters used by MiniV RNAP are characterized by conserved sequences and a 5-bp stem, 3-base loop hairpin structure.3,4

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 pmol of MiniV RNA Polymerase, 0.75 mM of each NTP and 20 ng/µl of SSB 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 Kit); lane 2, the expected 43-base RNA transcript produced.
P1 3´;-CAACGAAGCGTTGAATACC -5´;
P2 3´;-TTCTTCGAGGCGAAGAAAACC -5´;
P3 3´;-CGACGAGGCGTCGAAAACC-5´;

Figure 2A. Promoters recognized by N4 MiniV™ RNA Polymerase. Promoter P2 is the most characterized of the three and recommended for those constructing their own MiniV transcription single-stranded DNA templates.

3´-.... TTCTTCGAG GCGAAGAAAA CC ..... -5´ DNA template
+1 line  
5´- G ..... -3´ RNA transcript

Figure 2B. Transcription of RNA from a single-stranded DNA template containing the P2 promoter of phage N4.

References

  1. Kazmierczak, K.M., et al. (2002) EMBO J.21, 5815.
  2. Davydova, E.K. and Rothman-Denes, L.B. (2003) Proc. Natl. Acad. Sci. USA100, 9250.
  3. Haynes, L.L. and Rothman-Denes, L.B. (1985) Cell 41, 597.
  4. Glucksmann, M.A. et al. (1992) Cell 70, 491.

*Covered by issued and/or pending patents.

 

Catalog No. Size

MiniV™ In Vitro Transcription Kit
MV41025 25 Reactions
Contents: MiniV™ RNA Polymerase (contains an RNase inhibitor), MiniV™ 5X Transcription Buffer, E. coli Single-Stranded DNA Binding Protein, 10 mM ATP, CTP, GTP and UTP Solutions, DTT, RNase-Free Water, MiniV™ Control ssDNA Template, RNase-Free DNase I.

 

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