• Basic RT-PCR Kit using AMV RTase XL

Description

RNA PCR Kit Ver.3.0 is a basic RT-PCR Kit designed to amplify cDNA resulted from reverse transcription of RNA to cDNA, using AMV (Avian Myeloblastosis Virus) Reverse Transcriptase and TaKaRa Ex Taq™ HS in a single tube. The kit is composed of reagents necessary for reverse transcription and subsequent cDNA amplification, facilitating simplicity and efficiency in RNA analysis. Taking advantages of AMV RTase XL that are higher thermostability and broader range of reaction temperature (42 to 60°C) than RTase originated from MMLV, the kit allows high temperature transcription to prevent nonspecific stoppage due to secondary structure of RNA. The supplied Oligo dT-Adaptor Primer is designed to allow more efficient cDNA synthesis from 3′-terminus of poly(A)⁺ RNA. This enables amplification of unknown 3′-terminus utilizing 3′-RACE System. Also with this kit, RNA sample volume of maximum 9.5 µl can be applied in 20 µl-scale reverse transcription, which enables the use of low expression level RNA.
TaKaRa Ex Taq™ HS, which is a Hot Start PCR enzyme, is used for amplification to prevent non-specific amplification due to mispriming and primer dimerization before cycling that may occur when preparing reaction solutions.

Storage

-20°C

Kit components (100 reactions*)

AMV Reverse Transcriptase XL** (5 units/µl)	50 µl
Rnase Inhibitor (40 units/µl)	25 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
10× RT Buffer (100 mM Tris-HCl, pH8.3, 500 mM KCl)	120 µl
5× 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)	25 µl
Control F-1 Primer*** (20 pmol/µl) (upstream primer for positive control RNA)	25 µl
Positive Control RNA*** (2× 105 copies/µl) (transcribed poly(A)+ RNA of pSPTet3 plasmid)	25 µl

* One reaction means 10 µl RT followed by 50 µl PCR.
** Manufactured by Life Sciences, Inc.
*** Primers and Positive Control RNA are the same as ones supplied in RNA LA PCR Kit Ver. 1.1 (Cat.# RR012A).

Features

1. Any RNA can be used as a template.
2. Amplification size of cDNA can be extended at least up to 5 kbp.
3. AMV Reverse Transcriptase XL is employed to carry out transcription around 42 to 60°C.
4. TaKaRa Ex Taq™ HS decreases generation of non-specific PCR products, and the high specific PCR reaction is possible.
5. The primer for first strand cDNA synthesis can be appropriately selected from the following preparations: Random 9 mers and Oligo dT-Adaptor Primer are supplied in the kit. Specific downstream PCR primer can be prepared by the user.
6. Whole procedures are carried out in a single tube: After stopping reverse transcription with heat inactivation of enzyme, the reaction mixture for PCR is added into the tube and PCR is carried out continuously.
7. The components for 3′-RACE method are supplied in this kit. So it is available for 3′-RACE System by using Oligo dT-Adaptor Primer in reverse transcription, and by using M13 Primer M4 in PCR.