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StartScript® One Step RT-PCR Kit

StartScript® One Step RT-PCR Kit

Catalog Number: UA070126 Brand: UA BIOSCIENCE
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$75 USD
$75 USD
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Product Details

Product Specification


Stability & Storage

Store at -25 ~ -15℃ for 2 years
Reference


Components

Component

UA070126-50 Rxns

UA070126-250 Rxns

One-Step Enzyme Mix (25X)

100 μl

 5 × 100 μl

One-Step Reaction Mix (2X)

2 × 625 μl

10 × 625 μl

RNase Free dH2O

2 × 1 ml

10 × 1 ml


Protocol

1. Prepare the following mixture in an RNase-free microcentrifuge tube:

Component

Volume

One-Step Enzyme Mix (25X)

2 μl

One-Step Reaction Mix (2X)

25 μl

Gene-specific Forward Primer (10 μM)

2 μl

Gene-specific Reverse Primer (10 μM)

2 μl

Template RNA (1 pg~1 μg)

x μl

RNase-Free dH2O

Up to 50 μl
Mix gently by pipetting. 2. Perform the RT-PCR reaction under the following conditions:

Step

Temperature

Time

Number of Cycles

Reverse Transcription*

48°C

30 minutes

1 cycle

Initial Denaturation

94°C

3 minutes

1 cycle

Denaturation

Annealing**

Extension

94°C

50-65°C

72°C

30 seconds

30 seconds

1 minute

25–35 cycles

Final Extension

72°C

5 minutes

1 cycle

Soak

4°C

Indefinite

1 cycle
*If the template has complex secondary structures or high GC content, the reaction temperature can be increased to 55°C to improve yield. **The annealing temperature should be adjusted according to the primer's melting temperature (Tm), typically set 1–2°C below the primer Tm.

Guidelines

1. Treat all equipment used in the study with DEPC or purchase equipment certified to be nucleic acid-free. Wear gloves during the study and change them frequently to avoid RNase contamination. 2. Ensure that the reagents used are free of RNase contamination. 3. Typically, 28–30 cycles achieve optimal amplification. For low-copy target gene detection, the number of cycles can be increased to 40. 4. When performing reverse transcription reactions using this kit, specific reverse transcription primers must be used. Random Primer and Oligo dT Primer cannot be used. 5. Enzymes should be kept on ice during experimental operations and immediately stored at -20°C after the experiment.

Picture

Bioactivity

Using different amounts of HEK293T total RNA as templates, the DNA fragment of 197bp of the β-actin gene was detected. The electrophoresis results of RT-PCR amplification after adding a specified amount of this product or similar products from Company T (competitor) in a 50μl reaction system are shown in the figure. As shown in the figure, this product has a consistent detection sensitivity compared to Company T's products, and can sensitively detect RNA as low as 1pg, and has better specificity and amplification efficiency.
M: DL2000 DNA Marker；
CK: No Enzyme Mix added；
Lane 1: 1ug；
Lane 2: 100ng；
Lane 3: 10ng；
Lane 4: 1 ng；
Lane 5: 100pg；
Lane 6: 10pg；
Lane 7: 1pg；
Lane 8: 0.1 pg；