Store at -20°C,Avoid repeated freezing and thawing
Shipped In
Ice chest + Ice pads
Product Description
Product content:
M665754
Component
25 T
Storage
M665754A
Tris-HCl, 1 mM, PH 8.0
1 mL
-20℃. Avoid freeze/thaw cycle
M665754B
E. coli Poly(A) Polymerase, 5 U/μL
15 μL
-20℃. Avoid freeze/thaw cycle
M665754C
10×Poly(A) Polymerase Buffer
80 μL
-20℃. Avoid freeze/thaw cycle
M665754D
ATP, 10 mM
15 μL
-20℃. Avoid freeze/thaw cycle
M665754E
RT Primer, 25 μM
90 μL
-20℃. Avoid freeze/thaw cycle
M665754F
5×SuperRT Buffer
120 μL
-20℃. Avoid freeze/thaw cycle
M665754G
UltraPure dNTP Mix, 10 mM each
30 μL
-20℃. Avoid freeze/thaw cycle
M665754H
SuperRT, 200 U/μL
15 μL
-20℃. Avoid freeze/thaw cycle
M665754I
RNase-Free Water
1 mL
-20℃. Avoid freeze/thaw cycle
Product Introduction: This kit uses the method of adding a poly (A) tail at the 3 'end of miRNA to give miRNA a Poly (A) tail, followed by reverse transcription using Oligo (dT) - Universal tag universal reverse transcription primers to synthesize the first stranded cDNA corresponding to miRNA. The miRNA cDNA first strand synthesis kit contains all the reagents required for the miRNA 3 'end Poly (A) tail modification process and the reverse transcription process after modification. This kit has a very high Poly (A) modification and reverse transcription efficiency, which can range from 1 ng-2 μ The first strand of cDNA corresponding to miRNA was effectively obtained from the total RNA of g. And the operation is simple and fast, which can be used to simultaneously detect multiple miRNAs from a synthesized cDNA reaction. This not only reduces errors and saves samples, but also achieves high-throughput detection. Note: This kit must be used in conjunction with the miRNA fluorescence quantitative detection kit. Self prepared experimental materials: 1 ng-2 μ Total RNA of g, or 0.1 ng-1 μ Small molecule RNA of g.
Notes: To prevent RNase pollution, attention should be paid to the following aspects: 1. Use plastic products and gun heads without RNase to avoid cross contamination. 2. Glassware should be dry baked at a high temperature of 180 ℃ for 4 hours before use. Plastic containers can be soaked in 0.5 M NaOH for 10 minutes, thoroughly rinsed with water, and then sterilized under high pressure. 3. The solution should be prepared using water without RNase. 4. Operators should wear disposable masks and gloves, and change gloves frequently during the experiment.
Usage: A. The process of miRNA adding Poly (A) tail: 1.based on the amount of RNA used, dilute the total RNA of 10 mM ATP with 1 mM Tris (pH 8.0) according to the following formula: ATP dilution coefficient=5000/__ ng Example: If the initial amount of total RNA is 100 ng, then the ATP dilution coefficient is 5000/100=50. About to dilute ATP 50 times (1 μ 10 mM ATP plus 49 for l μ 1 mM Tris at pH 8.0. 2. Add the following reagents to the pre cooled RNase free reaction tube in the ice bath to a total volume of 25 μ L.
reagent
25 μlReaction system
final concentration
total RNA*
X μl
Up to 2 μg
10×Poly(A) Polymerase Buffer
2.5 μl
1×
Diluted ATP in step "1"
1 μl
/
E. coli Poly(A) Polymerase, 5U/μl
0.5 μl
2.5 U
RNase-Free Water
up to 25 μl
/
*The total RNA used in the reaction must contain small molecule RNA.
This process can also directly use small molecule RNA (recommended dosage of 2-5) μ L. Please determine the amount added based on the abundance of the target miRNA. 3. Gently mix the above reaction solution and briefly centrifuge to collect the liquid at the bottom of the tube. Incubate at 37 ℃ for 15 minutes. After this process is completed, immediately proceed with the synthesis of the first strand cDNA or temporarily store it at -20 ℃. If long-term storage is required, it is recommended to store at -80 ℃. B. The process of synthesizing the first strand of modified miRNA cDNA: 1. Add the reagents in the table below to the pre cooled RNase free reaction tube in the ice bath until the final volume reaches 20μl:
reagent
20 μlReaction system
The above Poly (A) reaction solution
4 μl
UltraPure dNTP Mix ，10 mM each
1 μl
RT Primer ，25 μM
3 μl
5×SuperRT Buffer
4 μl
SuperRT ，200 U/μl
0.5 μl
RNase-Free Water
7.5 μl
2. Gently mix the above reaction solution and briefly centrifuge to collect the liquid at the bottom of the tube. Incubate at 42 ℃ for 50 minutes. 3.85 ℃ for 5 minutes and terminate the reaction. The synthesized cDNA reaction solution can be directly used for fluorescence quantitative detection experiments or stored at -20 ℃ for future use.
