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Home > Products > PCR Products > RT-PCR and RACE > RNA PCR Kit, Version 3.0 Details

RNA PCR Kit, Version 3.0

Applications:

  • RT-PCR for analysis of gene expression at the RNA level in a conventional two-step, single-tube configuration

Description:

The RNA PCR Kit (AMV) Version 3.0 is designed for performing single tube reverse transcription of RNA to cDNA using AMV (Avian Myeloblastosis Virus) Reverse Transcriptase, with subsequent cDNA amplification using Takara Ex Taq™ HS. Taking advantage of AMV RTase XL, which possesses higher thermostability and a broader range of reaction temperatures (42–60°C) than MMLV RTase, this kit allows transcription at higher temperatures, which eliminates potential RNA secondary structure that can give rise to polymerase pauses. The supplied Oligo dT-Adaptor Primer is designed for highly efficient cDNA synthesis from the poly(A)+ RNA 3'-terminus, which allows amplification of unknown 3'-termini using 3'-RACE. The components for the 3'-RACE method are supplied in the kit. Finally, reverse transcription of low expression level RNA is also possible due to a 20 μL reverse transcription reaction in which a maximum RNA sample volume of 9.5 μL can be added. Takara Ex Taq™ HS (a high fidelity, hot start PCR polymerase), is used as the enzyme for cDNA amplification. This enzyme prevents non-specific DNA amplification due to mispriming and primer dimerization that may occur when preparing reaction solutions prior to cycling. Amplification of cDNA up to 5 kb in length is possible.

Kit Components:

TAK 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 Buffer 1 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: The original primer includes dT and complementary region 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 ones supplied in the RNA LA PCR Kit

Storage:

–20°C

Limited Use Label License: [P1][L15][M57]

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. 157:285-9.
  3. Frohman, M. A. et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998-9002./ol>

Figure Thumbnails:

RNA PCR Kit, Version 3.0 TAK RR019A 100 reactions 333.00
RNA PCR Kit, Version 3.0 TAK RR019B 200 reactions (A x 2) 650.00
Product Name Product Number Size Price Qty Add

*All prices in USD