| Product Description | This product is a reagent kit for reverse transcription of genomic DNA, which can remove genomic DNA in 2 minutes at 42 ℃. Meanwhile, the reverse transcription reagent contains components that inhibit gDNA Remover, and the samples treated with gDNA Remover can be directly subjected to reverse transcription reaction to synthesize cDNA.
This reagent kit is equipped with a novel and efficient reverse transcriptase HiFiScript, which significantly enhances the transcriptional activity of the enzyme through novel mutation sites. Meanwhile, reverse transcription reaction only takes 15 minutes to complete the synthesis of the first strand of cDNA. 5 × HifiScript RT MasterMix is a reverse transcription premix that contains all the reagents required for reverse transcription, making it easy and fast to operate. | H665909 | Component | 100 T | Storage | | H665909A | 10×gDNA Remover Mix | 100 µL | -20℃. Avoid freeze/thaw cycle. | | H665909B | 5×HiFiScript RT MasterMix | 400 µL | -20℃. Avoid freeze/thaw cycle. | | H665909C | RNase-Free Water | 1.5 mL | -20℃. Avoid freeze/thaw cycle. |
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Product features
1. Quick genome removal: gDNA Remover containing genomic DNA can remove genomic DNA in just 2 minutes.
2. Rapid reverse transcription: cDNA first strand synthesis can be completed in 15 minutes.
3. Convenient and fast: Ready to use reverse transcription Mix, easy to operate.
4. High sensitivity: cDNA first strand can be synthesized using pg level total RNA or mRNA templates.
5. Efficient reverse transcription efficiency: Novel mutation sites significantly enhance enzyme activity performance, resulting in higher yields of cDNA. Matters needing attention
1. During the operation, RNase contamination should be avoided to prevent RNA degradation or cross contamination during experiments. It is recommended that operators wear masks and disposable gloves, frequently change gloves, and use specialized instruments and consumables.
2. Disposable plastic containers should be used as much as possible for experiments. If glassware is used, a 0.1% DEPC (diethyl carbonate 1. ester) aqueous solution should be treated at 37 ℃ for 12 hours, and then sterilized under high pressure at 120 ℃ for 30 minutes before use. Alternatively, glassware should be sterilized under dry heat at 180 ℃ for 60 minutes before use. The sterile water used in the experiment should be treated with 0.1% DEPC and then subjected to high-pressure sterilization.
3. The reverse transcription system is prepared on ice for operation to prevent RNA degradation. The enzyme in the reagent kit should be stored at -20 º C as soon as possible after use, and repeated freeze-thaw should be avoided as much as possible. Usage
Thaw the template RNA on ice; After thawing at room temperature, immediately place the components of the reagent kit on ice. Before use, mix each solution by vortex oscillation and centrifuge briefly before use.
1、 Genomic DNA removal reaction
1. Prepare a reaction system on ice according to the following table, with a total volume of 10 μ L. To ensure the accuracy of the preparation of the reaction solution, a pre mixed system is first prepared in the amount of reaction number+2, then divided into each reaction tube, and finally RNA samples are added.
| Reagent |
10 μl Reaction system |
| 10×gDNA Remover Mix |
1µl |
| RNA Template¹ |
10 pg-1 μg |
| RNase-Free Water |
up to 10 µl |
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Note: 1) If the total RNA amount is greater than 1 µ g, please expand the reaction system proportionally.
2. Vortex shake and mix well, briefly centrifuge to collect the solution on the pipe wall to the bottom of the pipe.
3. Incubate at 42 ℃ for 2 minutes (during room temperature reaction, it can be extended to 30 minutes).
4. After the reaction is complete, centrifuge briefly and cool on ice.
2、 Reverse transcription reaction
1. Prepare the reaction system on ice according to the following table, and prepare the reaction solution on ice. To ensure the accuracy of the reaction solution configuration, first prepare a premixed solution in the amount of reaction number+2, and then divide it into 10 batches μ Take 10% of the prepared premix from each reaction tube μ Add to the reaction tube of step 1 that has completed genome removal.
| Reagent |
20 μl Reaction system |
| Step 1 Reaction solution |
10µl |
| 5×HiFiScript RTMaster Mix |
40µl |
| RNase-Free Water |
60µl |
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2. Mix well and centrifuge briefly to collect the solution on the tube wall to the bottom of the tube.
3. cDNA synthesis reaction conditions: Incubate at 37 ℃ for 15 minutes and 85 ℃ for 5 seconds.
4. After the reaction is completed, centrifuge briefly and place it on ice before proceeding with the subsequent reaction. If it needs to be stored for a long time, please place it at -20 ℃.
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