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Products >  PCR_Products >  RT-PCR-Two_Step >  AMV_Reverse_Transcriptase >  RNA_PCR_Kit

Two-Step RT-PCR

Takara's Two-Step RT-PCR kit, RNA PCR Kit (AMV) Version 3.0, is designed to perform single tube reverse transcription reactions using the AMV Reverse Transcriptase for cDNA synthesis. Subsequent cDNA amplification takes advantage of the properties of Takara Ex Taq HS, a high fidelity, hot-start DNA polymerase. 

The Takara RNA PCR kit uses AMV RTase XL, which possesses higher thermostability and a broader range of reaction temperatures (42–60°C) than MMLV RTase. This allows transcription to occur at higher temperatures and helps eliminate potential RNA secondary structures which can impede downstream cDNA amplification. The supplied Oligo dT-Adaptor Primer is designed for highly efficient cDNA synthesis from the poly(A)+ RNA 3'-terminus, allowing later amplification of unknown 3'-termini using 3'-RACE. Components for 3'-RACE are supplied in this two-step RT-PCR kit.

Reverse transcription of RNA expressed at a low level (rare RNAs) may also be performed by creating a 20 µL reverse transcription reaction mixture consisting of up to 9.5 µL RNA sample volume. Takara Ex Taq HS is the enzyme of choice for cDNA amplification because the Takara Ex Taq HS enzyme prevents non-specific room temperature DNA amplification as a result of mispriming and primer dimerization. Amplification of cDNAs up to 5 kb in length is possible using this RNA PCR kit.

At-A-Glance Documents Images & Data Resources

Applications

  • Two-step RT-PCR
  • cDNA synthesis

Components

RR019A

AMV Reverse Transcriptase XL* (5 U/µL)50 µL
RNase Inhibitor (40 U/µL)60 µL
Random 9-mers (50 pmol/µL) 50 µL
Oligo (dT) Adaptor primer (2.5 pmol/µL)50 µL
RNase-Free dH2O 1 mL
TaKaRa Ex Taq HS (5 Units/µL) 25 µL
M13 primer M4† (20 pmol/µL) 50 µL
10X RT Buffer (100 mM Tris-HCl, pH 8.3, 500 mM KCl) 1 mL
5X PCR Buffer1 mL
dNTP Mixture (ea. 10 mM) 150 µL
MgCl2 (25 mM) 1 mL
Control R-1 primer † (20 pmol/µL)25 µL
(downstream primer for positive control RNA)
Control F-1 primer † (20 pmol/µL)25 µL
(upstream primer for positive control RNA)
Positive control RNA ‡ (2 x 105 copies/µL)25 µL
(transcribed poly(A)+ RNA of pSPTet3 plasmid)

* Manufactured by Life Science Co.
‡ Positive control RNA: SP6 RNA polymerase was used to transcribe the supplied control RNA in vitro from plasmid pSPTet3, which contained a cloned 1.4 kb tetracycline resistance gene from pBR322. The control RNA is a poly(A+) RNA with a 30 base poly(A) tail. Plasmids cloned with a full-length double-stranded cDNA prepared from this control RNA show tetracycline resistance.

Primer

† Primer Sequences: Random 9 mers: 5'-NNNNNNNNN-3'; Oligo dT-Adaptor Primer: Includes dT and region complementary to M13 primer M4; Control F-1 primer: 5'-CTGCTCGCTTCGCTACTTGGA-3'; Control R-1 primer: 5'-CGGCACCTGTCCTACGAGTTG-3'; M13 primer M4: 5'-GTTTTCCCAGTCACGAC-3'
‡ Primers and Positive Control RNA are the same as those supplied in the RNA LA PCR Kit.

Storage

–20°C

References

  1. Kawasaki, E. S. and Wang, A. M. (1989) PCR Technology (Erlich, H. K. ed.), Stockton Press: 89–97.
  2. Lynas, C. et al. (1989) J. Pathol. 157:285–289.
  3. Frohman, M. A. et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998–9002.



 
 
Products
Cat. # Product Package Size Price License # of Units Select
RR019A RNA PCR Kit (AMV) Version 3.0 100 Rxns $353.00 License Statements
RR019B RNA PCR Kit (AMV) Version 3.0 200 Rxns $690.00 License Statements
 

 

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