High Efficient Reverse Transcriptase
Code No. TRT-101 10,000U
ReverTra Ace™ is a high efficient M-MLV (Moloney Murine Leukemia Virus) reverse transcriptase that has been genetically modified to remove RNase H activity and increase reaction efficiency. It is the preferred enzyme for applications requiring full-length cDNAs and high product yields from total RNA, mRNA, rRNA, etc.
Fig. 1. Point mutation in M-MLV RTase
E. coli strain carrying a cloned, modified M-MLV reverse transcriptase gene.
One unit is defined as the amount of enzyme required to incorporate 1 nmole of dTTP into an acid-insoluble material in 10 min at 42ºC.
50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 10 mM DTT, 0.01% Nonidet™ P-40,
50% Glycerol. Store at -20ºC
ReverTra Ace™ (100U/µL)
5 x Buffer
Component | Amount | |
---|---|---|
RNA | Total RNA Poly (A)+ RNA |
0.1-1 µg 50-500 ng |
Primer | Oligo (dT) Random Gene specific |
5 pmoles 25 pmoles 5 pmoles |
5 x Buffer | 4 µL | |
10 mM dNTPs* | 2 µL | |
(RNase inhibitor) | 20 U | |
Nuclease-free water | X µL | |
Total reaction volume | 20 µL |
* This reagent is not supplied with this product.
30ºC, 10 min**
42ºC, 20-60 min
99ºC, 5 min
** when using random primers
Component | Amount |
---|---|
Poly (A)+ RNA | 50-500 ng |
Oligo (dT) primer | 5 pmoles |
5 x Buffer | 4 µL |
10 mM dNTPs* | 2 µL |
RNase inhibitor | 20 U |
Nuclease-free water | X µL |
Total reaction volume | 20 µL |
* This reagent is not supplied with this product.
42ºC, 30 min
99ºC, 5 min
cDNAs were synthesized with oligo (dT)30 primers and 100 U enzyme/poly (A)+ RNA mixture (1.35-9.49 kb, 0.4 mg) as templates for 30 min at various temperatures. cDNAs were labeled with [32P-dCTP] during the reaction. The synthesized cDNAs were separated by 1% denatured agarose gel electrophoresis, and detected. The results suggested that ReverTra Ace™ can elongate efficiently at 42-55ºC compared to other RNase H minus RTases from other companies.
G3PDH genes (500 bp) were amplified by PCR using cDNA templates that were synthesized with various RNase H minus RTases from G3PDH mRNA (102-105 copies/reaction). The RTase reaction was performed with specific reverse primers and 100 U enzyme at 42ºC for 20 min. The results indicated that ReverTra Ace™ is suitable for RT-PCR amplifications that require sensitivity.
cDNA was synthesized by ReverTra Ace™ using a specific primer for the 3’-end of dystrophin mRNA at 42ºC for 30 min. The 5’ region at a distance of 14 kb from the 3’ end of the dystrophin gene was amplified by PCR. The result indicated that ReverTra Ace™ can elongate cDNA of ≥ 14 kb.