Description
Product description
This product, based on the classic reverse transcription reagents, has optimized the reaction time. The total reaction time can be as short as less than 6 minutes, while maintaining stable detection rates, specificity, and yield. It is suitable for reverse transcription of RNA templates that are complex, present in low amounts, or represent low-copy genes.
The reverse transcription product of this product can be used for downstream PCR or qPCR applications. The kit provides two types of cDNA synthesis primers: Random Primers N6 and Oligo (dT)18. Users can choose according to their needs.
Specifications
|
Catalog Number
|
N132064E
|
N132064S
|
|
Specifications
|
10 T
|
100 T
|
Components
|
Catalog Number
|
Component Name
|
N132064E
|
N132064S
|
|
N132064-A
|
4×Fast cDNA Synthesis Mix (No Dye)
|
50 μL
|
500 μL
|
|
N132064-B
|
5×gDNA Digester Mix
|
20 μL
|
200 μL
|
|
N132064-C
|
Random Primers（50 μM）
|
20 μL
|
200 μL
|
|
N132064-D
|
Oligo d(T)18 Primers (50 μM)
|
20 μL
|
200 μL
|
|
N132064-E
|
RNase-free H2O
|
200 μL
|
2×1 mL
|
Storage
Store at -25 to -15℃. Valid for 1 year.
Notes
1. All operations should be carried out on ice, and RNase contamination should be avoided during the process.
2. For your safety and health, please wear a lab coat and disposable gloves when operating.
3. For Research Use Only.
Instructions
1. Reverse Transcription with gDNA Removal Step
1) gDNA Digestion
Prepare the following mixture in an RNase-free centrifuge tube and gently mix by pipetting. Incubate at 42°C for 2 minutes.
|
Component
|
Usage Amount
|
|
5×gDNA Digester Mix
|
2 μL
|
|
Total RNA or mRNA
|
Total RNA：10 pg-5 μg
mRNA：10 pg-500 ng
|
|
RNase-free H2O
|
Up to 10 μL
|
【Note】It is recommended that the input amount of Total RNA does not exceed 2 µg. If the expression level of the target gene is low, the input amount can be increased up to a maximum of 5 µg.
2) Reverse Transcription Reaction Setup
|
Component
|
Usage Amount
|
|
The reaction mixture from the previous step
|
10 μL
|
|
4×Fast cDNA Synthesis Mix (No Dye)
|
5 μL
|
|
Oligo d(T)18 Primers（50 μM) or Random Primers（50 μM）
|
2 μL
|
|
RNase-free H2O
|
Up to 20 μL
|
【Notes】
a. The amount of primer can be adjusted according to the amount of template input. If the downstream experiment is qPCR, Random Primers can be added to the reaction mixture at the recommended amount.
b. It is recommended to first add the 4×Fast cDNA Synthesis Mix (No Dye) and mix thoroughly before adding the reverse transcription primer to ensure that the primer is not affected by the gDNA Digester.
3) Reverse Transcription Procedure Settings
|
Temperature
|
Time
|
|
55℃
|
5 min
|
|
85℃
|
5 sec
|
2. Reverse Transcription without gDNA Removal Step
1) Reverse Transcription Reaction Setup
|
Component
|
Usage Amount
|
|
4×Fast cDNA Synthesis Mix (No Dye)
|
5 μL
|
|
Oligo d(T)18 Primers（50 μM) or Random Primers（50 μM）
|
2 μL
|
|
Total RNA or mRNA
|
Total RNA：10 pg-5 μg
mRNA：10 pg-500 ng
|
|
RNase-free H2O
|
Up to 20 μL
|
2) Reverse Transcription Procedure Settings
|
Temperature
|
Time
|
|
55℃
|
5 min
|
|
85℃
|
5 sec
|
【Notes】
a. It is recommended that the input amount of Total RNA does not exceed 2 µg. If the expression level of the target gene is low, the input amount can be increased up to a maximum of 5 µg.
b. The amount of primer can be adjusted according to the amount of template input. If the downstream experiment is qPCR, Random Primers can be added to the reaction mixture at the recommended amount.
c. For templates with high GC content or complex structures, the reverse transcription temperature can be increased to 60°C.
d. This product can synthesize cDNA sequences up to 14 kb in length. If longer cDNA products are required, the reverse transcription time can be appropriately extended.
