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Reverse Transcriptases

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SuperiorScript III Reverse Transcriptase

No Product Name Cat.# Conc Size Price(₩) Add
1 SuperiorScript III Reverse Transcriptase RT006M 200 units/μl 10,000 units ₩ 190,000 추가
2 SuperiorScript III Reverse Transcriptase RT006L 200 units/μl 50,000 units ₩ 760,000 추가
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Product description

SuperiorScript III Reverse Transcriptase is genetically engineered version of M-MLV RT which is reduced RNase H activity and increased thermostability, thus this enzyme can synthesize first-strand cDNA at elevated temperatures up to 55 . SuperiorScript III Reverse Transcriptase shows improved cDNA yields and RNA detection sensitivity than the competitor’s enzyme. This enzyme is capable of synthesizing cDNA from 100 bp to 12 kb or more.

 

Applications

- Synthesis of first-strand cDNA,

- Array labeling

- cDNA library construction

- 3’ and 5’ RACE, RT-PCR

- Primer extension

 

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 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% NP40, 50% glycerol

 

5X First-Strand Buffer

250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15mM MgCl2

 

Standard reaction conditions

5X First-Strand buffer

4 μl

SuperiorScript III Reverse Transcriptase (200 units/μl)

1 μl

dNTP Mixture (10 mM each)

1 μl

0.1 M DTT

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 37 for 5 min.

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

→Incubate at 50 for 30~60 min.

    (Increase the reaction temperature to 55°C for gene-specific primer.)

→Incubate at 70 for 15 min to inactivate the reaction.




Figure 1. Thermostability of SuperiorScript III on 3 targets

cDNA was synthesized from 500 ng total HeLa RNA for beta-actin 184 bpGAPDH 395 bp and TFR 738 bp. One-tenth of the cDNA reaction was used for 30 cycles of PCR with nTaq-multi HOT (Cat.# P725HC).




Figure 2. Sensitivity of SuperiorScript III

cDNA was Synthesized from HeLa total RNA with oligo(dT) primer using buffer supplied with each RT and recommended conditions. 10% of cDNA reaction was added to 20 μPCR reaction containing primers for 500 bp GAPDH gene and 2X TOPsimple™ DyeMIX-HOT (Cat.# P510H), 30 cycle1 min/kb.




Figure 3. High sensitivity using SuperiorScript III for RT-qPCR

cDNA was synthesized using SuperiorScript III from 100 ng to 0.1 ng total HeLa RNA for GAPDH 157 bp. cDNA was amplified using TOPreal™ qPCR 2X PreMIX (SYBR Green with low ROX) (Cat.# RT500) on the Bio-Rad CFX-96.

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