- cDNA synthesis kits
- Reverse transcriptases
- RACE kits
- Purified cDNA & genomic DNA
- Purified total RNA and mRNA
- cDNA synthesis accessories
The RNA PCR Kit (AMV) Version 3.0 combines AMV Reverse Transcriptase with Takara Ex Taq HS—a high-fidelity, hot-start DNA polymerase—enabling users to synthesize and amplify cDNA in a single tube, using a two-step protocol. The RNA PCR kit uses AMV RTase XL, which possesses higher thermostability and a broader range of reaction temperatures (42–60°C) than MMLV RT. This allows transcription to occur at higher temperatures and helps eliminate potential RNA secondary structures that 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.
- Two-step RT-PCR
- cDNA synthesis
|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 U/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)
(downstream primer for positive control RNA)
|Control F-1 primer† (20 pmol/µl)
(upstream primer for positive control RNA)
|Positive control RNA** (2 x 105 copies/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 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.
Reverse transcription of RNA expressed at low levels (rare RNAs) may also be performed by creating a 20 µl reaction mixture with an RNA sample volume of up to 9.5 µl. Takara Ex Taq HS is the enzyme of choice for cDNA amplification because it prevents non-specific room temperature DNA amplification due to mispriming and primer dimerization. Amplification of cDNAs up to 5 kb in length is possible using this RNA PCR kit.
Frohman, M. A., Dush, M. K. & Martin, G. R. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. U. S. A. 85, 8998–9002 (1988).
Kawasaki, E. S., and Wang, A. M. PCR Technology: Principles and Applications for DNA Amplification. (Palgrave Macmillan UK, 1989).
Lynas, C., Cook, S. D., Laycock, K. A., Bradfield, J. W. & Maitland, N. J. Detection of latent virus mRNA in tissues using the polymerase chain reaction. J. Pathol. 157, 285–9 (1989).
Additional product information
Please see the product’s Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.
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