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TOPscript™ II Reverse Transcriptase (RNase H-)

No Product Name Cat.# Conc Size Price(₩) Add
1 TOPscript™ II Reverse Transcriptase (RNase H-) RT003S 200 units/μl 10,000 units ₩ 120,000 추가
2 TOPscript™ II Reverse Transcriptase (RNase H-) RT003M 200 units/μl 20,000 units ₩ 216,000 추가
3 TOPscript™ II Reverse Transcriptase (RNase H-) RT003L 200 units/μl 50,000 units ₩ 480,000 추가
4 TOPscript™ II Reverse Transcriptase (RNase H-) RT003H 1000 units/μl 50,000 units ₩ 480,000 추가
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Product description

TOPscript™ II Reverse Transcriptase (RNase H-) is genetically engineered recombinant protein that possesses moderate thermostability (42~55) and lacks RNase H activity. TOPscript™ II Reverse Transcriptase (RNase H-) does not degrade RNA strands from RNA-DNA hybrids and therefore is capable of high yield of synthesizing cDNA. The optimum temperature of TOPscript™ II Reverse Transcriptase (RNase H-) is 50. TOPscript™ II Reverse Transcriptase (RNase H-) does not show inhibitory effect on one-step RT-PCR reaction.

 

Characteristics

- Themostable and RNase H variant of M-MLV RTase (RTase H-)

- Synthesize cDNA at 42~55

- Especially the highest activity at 50

- Synthesis of long length cDNA (14 kb)

- Efficiently synthesize cDNA by inhibiting the formation of secondary structure of RNA

 

Applications

- First-strand cDNA synthesis

- RT-PCR

- Real-Time PCR

- Primer extension analysis

 

Quality control

- Purity: >99% on SDS-PAGE

- Endonuclease-free

- Exonuclease-free

- RNase-free

- Inhibitor-free

- Yield and length of cDNA product

 

Unit definition

One unit is the amount of enzyme required to incorporate 1 nmol dTTP into acid-insoluble materials using 0.4 mM poly(rA)-oligo(dT) as substrate at 37°C in 10 min.

 

Supplied with

- 10X TOPscript RT Buffer

- dNTP Mixture (2 mM each)

- Sterile water (RNase free)

 

Storage buffer

20 mM Tris-Cl (pH7.5), 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.01% NP40, 50% glycerol

 

10X TOPscript™ RT Buffer

500 mM Tris-HCl (pH 8.3), 30 mM MgCl2, 100 mM DTT, 750 mM KCl

 

Standard reaction conditions

10X TOPscript™ RT Buffer

2 μl

TOPscript™ II Reverse Transcriptase (200 units/μl)

1 μl

dNTP Mixture (2 mM each)

2 μl

a)Template RNA

X μl

b)Primer

1 μl

RNase Inhibitor (40 units/μl)

0.5 μl

Sterile water (RNase free)

up to 20 μl

a)Prepare one of the following RNA template.

    - Total RNA: 1 ng~5 μg

    - Messenger RNA (mRNA): 1 ng~250 ng

    - Specific RNA: 0.01 pg~0.5 μg

b)Prepare one of the following primers.

    - Oligo (dT)18: 50 μM~100 μM

    - Random hexamer: 50 μM~100 μM

    - Specific primer: 15 pmol~20 pmol

→An additional annealing step is recommended:

    - if using oligo(dT)18, incubate at 42 for 5 min.

    - if using random hexamer, incubate at 25 for 10 min.

→Incubate at 42~60 for 60 min.

→Incubate at 95 for 5 min to inactivate the reaction.

 

Note

TOPscript™ Reverse Transcriptase performs optionally over the full range of 42-60. Typically, 50 is a good starting point. For RNAs containing secondary structure and other challenging targets, a synthesis temperature of 60 may be used without loss of performance.

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