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Reverse Transcriptases

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TOPscript™ cDNA Synthesis Kit

No Product Name Cat.# Conc Size Price(₩) Add
1 TOPscript™ cDNA Synthesis Kit EZ005S - 50 reactions ₩ 250,000 추가
2 TOPscript™ cDNA Synthesis Kit EZ005M - 100 reactions ₩ 450,000 추가
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Product description

TOPscript cDNA Synthesis Kit is designed for the highest efficiency conversion of RNA to cDNA. TOPscript Reverse Transcriptase is genetically engineered version of M-MLV Reverse Transcriptase which is highly thermostable, thus can synthesize cDNA at elevated temperatures up to 60 A two-step RT-PCR format is useful for amplifying multiple targets from a single cDNA source, for maintaining archival cDNA, and for providing maximum flexibility in selecting a downstream qPCR reagent system. The TOPscript cDNA synthesis Kit is formatted for high-yield production of cDNA up to 20 kb and each reaction accommodates a range of RNA amounts from fg to μg.

 

Characteristics

- Robust cDNA synthesis across a broad range of temperatures from 42~60

- Produces excellent yields of amplifiable cDNA

- Can synthesize long cDNA targets, up to 20 kb, from small amounts of input RNA

- Able to reverse transcribe through RNA secondary structures

- Strict enzyme purity specifications

 

Applications

- cDNA cloning or cDNA library preparation

- Two step RT-PCR (end-point or real-time quantitative PCR)

 

Supplied with

- TOPscript™ Reverse Transcriptase (200 U/μl)

- 10X TOPscript™ RT Buffer

- RNase Inhibitor (40 U/μl)

- dNTP Mixture (2 mM)

- Oligo dT (80 μM)

- Random hexamer (100 μM)

- Sterile water (RNase free)

 

Quality control

- Purity: >99% on SDS-PAGE

- Endonuclease-free

- Exonuclease-free

- RNase-free

- Inhibitor-free

- Satisfactory yield and length of cDNA products

 

Unit definition

One unit is defined as the amount of enzyme required to incorporate 1 nmol of dTTP into acid-insoluble materrials using 0.4 mM poly(rA)-oligo(dT) as substrate at 37 in 10 min in a 1X reaction buffer.

 

Standard reaction conditions

10X TOPscript™ RT Buffer

2 μl

TOPscript™ 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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