제품 설명TOPscript™ Reverse Transcriptase는 M-MLV Reverse Transcriptase(Cat. RT001) 의 개량형 제품입니다. 이 제품은 높은 열 안정성 (~60°C)과 RNase H 활성이 없는 제품으로 최대 20kb까지 cDNA 합성이 가능합니다. 해당 제품은 Primer (oligo dT, random hexamer) 별도 구매가 필요 합니다.
제품 특징
- Molecular weight: 71 kDa
- Broad reaction temperature: 37℃~60℃
- Synthesis of long cDNA
- Excellent sensitivity
제품 응용
- Synthesis of first-strand cDNA,
- Array labeling
- cDNA library construction
- 3’ and 5’ RACE, RT-PCR
- Primer extension
제품 구성품
- TOPscript™ Reverse Transcriptase
- 10X TOPscript™ RT Buffer
- dNTP Mixture (2 mM each)
- Sterile water (RNase free)
제품 품질 관리
- Purity: >99% on SDS-PAGE
- Endonuclease-free
- Exonuclease-free
- RNase-free
- Inhibitor-free
- Satisfactory yield and length of cDNA products
Unit의 정의
One unit is the amount of enzyme required to incorporate 1 nmol of dTTP into acid-insoluble materials using 0.4 mM poly(rA)-oligo(dT) as substrate at 37℃ in 10 min.
Storage buffer
20 mM Tris-Cl (pH7.5), 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.01% TritonX-100, 55% glycerol
주의 사항
TOPscript ™ 역전사효소는 42 ℃ -60 ℃의 넓은 범위에서 선택적으로 수행됩니다. 일반적으로 50 ℃를 권장합니다. 2 차 구조 및 기타 까다로운 표적을 포함하는 RNA의 경우, 성능 손실없이 합성 온도를 60 ℃로 할 수 있습니다.
Figure 1. TOPscript™ Reverse Transcriptase has wide optimal temperatures.
Synthesis of cDNA from mRNA encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was carried out by TOPscript™ Reverse Transcriptase (lanes 1-4) at 42, 50, and 60℃. The cDNA of GAPDH was also synthesized by other suppliers、reverse transcriptases (lanes 5-16) for comparison purposes. Amplification of the GAPDH cDNA was performed by nTaq-Tenuto DNA Polymerase (Cat.# P225 or P250).
Mw, 1 kb (+) DNA Ladder Marker (Cat.# DM003)
Figure 2. Efficient synthesis of cDNA from long mRNA by TOPscript™ Reverse Transcriptase
Total RNA (100 ng) isolated from HeLa cells were first converted into cDNA using oligo (dT) by either TOPscript™ Reverse Transcriptase (lanes 1, 3, and 5) or Supplier A’s reverse transcriptase (lanes 2, 4, and 6). Reactions were carried out at 42℃, 50℃, and 60℃ as indicated. Efficiency of LRP1 cDNA (14.9 kb) synthesis was verified by amplifying the 5’ end 0.9-kb region of the LRP1 cDNA using nTaq-Tenuto DNA Polymerase (Cat #. P225 or P250). Mw, 1kb (+) DNA Ladder Marker (Cat.# DM003).
