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Description
StartScript® One Step RT-PCR KitProduct 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 l 5 100 l One Step Reaction Mix (2X) 2 625 l 10 625 l RNase Free dH2O 2 1 ml 10 1 ml Protocol 1. Prepare the following mixture in an RNase free centrifuge tube: Component Volume One Step Enzyme Mix (25X) 2 l One Step Reaction Mix (2X) 25 l Gene specific Forward Primer (10 M) 2 l Gene
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 centrifuge 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 evenly by gently pipetting up and down.
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 minutes |
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 regions, the reaction temperature can be increased to 55°C, which helps improve the yield.
*The annealing temperature needs to be adjusted according to the primer annealing temperature, and it is generally set to be 1-2°C lower than the primer annealing temperature.
Guidelines
1. Treat all equipment used in the research with DEPC, or purchase equipment that is certified nucleic acid-free. Wear gloves during the research and change them frequently to avoid RNase contamination.
2. Ensure that there is no RNase contamination in the reagents used.
3.Under normal circumstances, 28-30 cycles can achieve optimal amplification; for the detection of low-copy target genes, 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 Primers and Oligo dT Primers cannot be used.
5. During experimental operation, enzyme products should be placed on ice, and immediately stored at -20°C after the experiment is completed.
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