Product Description
All in One RT MasterMix (with dsDNase) is an efficient, convenient, and high-quality single stranded cDNA synthesis kit, which includes thermally stable M-MLV GIII reverse transcriptase and its reaction buffer, RNAse inhibitors, dNTPs, Oligo (dT) 20VN, and random primers, all the necessary components for reverse transcription reaction. The reaction can be initiated by adding RNA template and water, and the generated cDNA is mainly used for subsequent qPCR analysis.
The extracted RNA samples often contain genomic DNA (gDNA) contamination, which may lead to simultaneous amplification of gDNA and cDNA in qPCR, affecting quantitative accuracy. This kit efficiently removes gDNA through dsDNase, specifically degrades DNA strands in double stranded DNA or DNA-RNA heterozygous chains, and rapidly and irreversibly inactivates them at reverse transcription temperature. Compared with the traditional use of DNase I, it does not require additional EDTA treatment, simplifies the experimental process, and reduces the risk of inhibition of reverse transcription reactions.
This kit can perform both reverse transcription and gDNA removal reactions in the same tube, with simple operation and effective reduction of the risk of sample contamination and RNA degradation caused by complex sample addition processes. But if the degree of genomic contamination is severe, it is recommended to choose a method that separates the removal of gDNA contamination from reverse transcription.
ordering information
product name | Item number | specifications |
All-in-One RT MasterMix (with dsDNase) | AN33L718 | 20T |
All-in-One RT MasterMix (with dsDNase) | AN33L719 | 100T |
Product components
components | specifications |
A. All-in-One RT MasterMix | 400 μL |
B. dsDNase | 2*50 μL |
C. 10× dsDNase Buffer | 200 μL |
D. Nuclease-Free Water | 2*1 mL |
Transportation and storage
Blue ice transportation- Stored at 20 ℃, with a shelf life of 24 months.
usage method
1. For RNA samples with low genomic DNA content (recommended solution)
① Prepare the following reaction system on ice:
reagent | usage |
模板 RNAa | 50 ng~1 μg |
All-in-One RT MasterMix | 4 μl |
dsDNase | 1 μl |
Nuclease-Free Water | To 20 μl |
It is recommended to use high-quality RNA extracted from the reagent kit as a template.
② Incubate at 37 ℃ for 2 minutes to remove gDNA contamination;
③ Incubate at 55 ℃ for 15 minutes;
④ After the reaction is complete, incubate at 85 ℃ for 5 minutes to terminate the reaction;
⑤ Quickly place the obtained cDNA on ice for subsequent experiments; Or immediately store at -20 ℃.
2. Targeting RNA samples with high genomic DNA content
(1) Genomic DNA contamination removal
① Prepare the following reaction system on ice:
reagent | usage |
模板 RNAa | 50 ng~1 μg |
dsDNase | 1 μl |
10× dsDNase Buffer | 1 μl |
Nuclease-Free Water | To 10 μl |
a. It is recommended to use RNA extracted from the reagent kit as a template.
② Gently suck, beat, mix well, and leave immediately;
③ Incubate at 37 ℃ for 2 minutes to remove genomic DNA contamination;
Note: If the genomic DNA contamination in RNA is severe, the incubation time at 37 ℃ can be appropriately extended to 5 minutes.
④ Incubate at 65 ℃ for 2 minutes to inactivate dsDNase and place on ice.
(2) First strand cDNA synthesis
① Prepare the following reaction system on ice:
reagent | Usage (experimental group) |
“实验 (1)”反应产物 | 10 μl |
All-in-One RT MasterMix | 4 μl |
Nuclease-Free Water | To 20 μl |
② Gently suck, beat, mix well, and leave immediately;
③ Incubate at 50 ℃ for 15 minutes;
Note: If the target RNA does not contain Poly (A) structure, it can be incubated at 25 ℃ for 10 minutes in advance.
④ After the reaction is complete, incubate at 85 ℃ for 5 minutes to terminate the reaction;
⑤ Place the obtained cDNA solution on ice for subsequent experiments.
Note: The cDNA solution should be stored at -20 ℃ for no more than one week; Can be stored for a long time at -80 ℃.
matters needing attention
1. The premix already contains Oligo (dT) 20VN and random primers, which are suitable for eukaryotic mRNA containing Poly (A) structure, as well as prokaryotic RNA, eukaryotic rRNA, tRNA and other templates without Poly (A) structure, but not for small RNA templates such as miRNA.
2. Due to the fact that random primers can initiate reverse transcription at any position of RNA, it is not recommended to use this product for cloning full-length cDNA in eukaryotes.
3. This product is only for scientific experimental research and should not be used in clinical diagnosis, treatment, or other fields.
This product is only for scientific experimental research and should not be used in clinical diagnosis, treatment, or other fields.
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