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TOPscript™ Reverse Transcriptase

No Product Name Cat.# Conc Size Price(₩) Add
1 TOPscript™ Reverse Transcriptase RT002S 200 units/μl 10,000 units ₩ 120,000 추가
2 TOPscript™ Reverse Transcriptase RT002M 200 units/μl 20,000 units ₩ 216,000 추가
3 TOPscript™ Reverse Transcriptase RT002L 200 units/μl 50,000 units ₩ 480,000 추가
4 TOPscript™ Reverse Transcriptase RT002H 1000 units/μl 50,000 units ₩ 480,000 추가
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Product description

TOPscript™ Reverse Transcriptase is genetically engineered version of M-MLV RT which is highly thermostable, thus can synthesize cDNA at elevated temperatures up to 60. This property is very useful when RNA templates are long and have extensive secondary structures. TOPscript™ Reverse Transcriptase is capable of synthesizing cDNA longer than 20 kb from messenger RNA.

 

Characteristics

- Molecular weight: 71 kDa

- Broad reaction temperature: 37~60

- Synthesis of long cDNA

- Excellent sensitivity

 

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.

 

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™ 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.



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 suppliersreverse 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 5end 0.9-kb region of the LRP1 cDNA using nTaq-Tenuto DNA Polymerase   (Cat #. P225 or P250). Mw, 1kb (+) DNA Ladder Marker (Cat.# DM003).


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