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Hifair® V Reverse Transcriptase

Product SKU

Specifications

Price

11300ES92

10000 U

$250.59

11300ES93

50000 U

$868.19

Product Description

Hifair® V Reverse Transcriptase is a new reverse transcriptase obtained by genetic engineering technology on the basis of Hieff® M-MLV (H- ) Reverse Transcriptase. Compared with Hieff® M-MLV (H- ) Reverse Transcriptase, its thermal stability is greatly improved. The enzyme is active up to 60 ℃ and suitable for reverse transcription of RNA template with complex secondary structure. And the enzyme enhances the affinity with the template, is suitable for reverse transcription of a small number of templates and low copy genes. The enzyme is providing higher specificity, higher yield of cDNA and more full-length cDNA product up to 10 kb.

Package Information

Component Number

Components

11300ES92 (10000 U)

11300ES93 (50000 U)

11300-A

Hifair® V Reverse Transcriptase (200 U/μL)

50 μL

250 μL

11300-B

5×Hifair® V Buffer

250 μL

1.25 mL

Applications

RT-qPCR, cDNA Synthesis, Reverse Transcription (cDNA Synthesis)

Unit Definition

One unit is defined as the amount of enzyme that will incorporate 1 nmol of dTTP into acid-insoluble material in 10 minutes at 37°C using Poly(A) .Oligo(dT) as template.

Quality Control

Exonuclease Activity: Incubation of 200 U of this enzyme with 1 μg λ-Hind III at 37℃ for 1 hour results in no change on electrophoretic bands.

Endonuclease Activity: Incubation of 200 U of this enzyme with 1 μg Supercoiled pBR322 DNA for 1 hour at 37℃ results in no change on electrophoretic bands.

Contamination of Rnase: Incubation of 200 U of this enzyme with 1 μg of mouse liver RNA for 1 hour at 37℃ results in no change on electrophoretic bands.

Notes

1. Keep the experiment area clean; Wear clean gloves and masks, and use new centrifuge tubes, tips and other supplies to ensure experiment is RNase free;

2. All operations should be carried out on ice to prevent RNA degradation;

3. High quality RNA samples are recommended for efficient reverse transcription;

4. For your safety and health, please wear experimental clothes and disposable gloves.

Protocol for cDNA Synthesis reaction

1. Denaturation of RNA template(This step is optional, denaturation of RNA template helps to open the secondary structures to improve the first strand cDNA yield.)

Components

Volume

RNase free ddH2O

to 13 μL

Oligo (dT)18 (50 μM)

1 μL

or Random Primers (50 μM)

or 1 μL

or Gene Specific Primers (2 μM)

or 1 μL

RNA template

Total RNA: 1 ng -5 μg or mRNA:1 ng-500 ng

Incubate at 65℃ for 5 minutes, place on ice rapidly and let rest for 2 minutes. Brief centrifugation to collect reaction liquid.

2. Preparation of a first strand cDNA synthesis reaction mixture(20 μL volume)

Components

Volume

Mixture of previous step

13 μL

5×Hifair® V Buffer

4 μL

dNTP Mix (10 mM)

1 μL

Hifair® V Reverse Transcriptase (200 U/μL)

1 μL

RNase inhibitor (40 U/µL)

1 μL

3. Perform the first strand cDNA synthesis reaction under the following conditions

Temperature

Time

25℃*

5 min

42℃**

15-30 min

85℃

5 min

*This step is required only when using the random hexamers. Please omit this step when using Oligo (dT)18 or Gene Specific Primer.

**For template with complicated secondary structures or high GC content, you can raise the reaction temperature to 50℃- 55℃to increase the cDNA yield.

The product can directly be used in PCR or qPCR reactions, or store at -20℃ for a short-term storage. It is recommended to aliquot and store at -80℃ for a long-term storage. Avoid repeated freezing and thawing.

15221439726

15221439726