| Product Description | This product is a cDNA first strand synthesis kit specially prepared for the first step experiment of two-step RT-PCR. This product contains all the reagents required for reverse transcription from RNA templates to cDNA first strand, including HiFi MMLV reverse transcriptase, reaction buffer, primers, dNTP, etc. The mutated HiFi MMLV reverse transcriptase RNase H activity is deficient, reducing RNA degradation in reverse transcription reactions and making it easier to obtain full-length cDNA. HiFi MMLV reverse transcriptase has strong thermal stability and can yield high yields of cDNA, making it simple and convenient to use. This system has high compatibility with subsequent PCR and quantitative PCR experiments, and is suitable for various DNA polymerase reactions.
| H665693 |
Component |
100 T |
Storage |
| H665693A |
HiFi-MMLV, 200 U/μL |
100 μL |
-20℃. Avoid freeze/thaw cycle. |
| H665693B |
5×RT Buffer |
500 μL |
-20℃. Avoid freeze/thaw cycle. |
| H665693C |
Primer Mix |
240 μL |
-20℃. Avoid freeze/thaw cycle. |
| H665693D |
dNTP Mix, 2.5 mM Each |
500 μL |
-20℃. Avoid freeze/thaw cycle. |
| H665693E |
DTT, 0.1 M |
240 μL |
-20℃. Avoid freeze/thaw cycle. |
| H665693F |
RNase-Free Water |
1 mL |
-20℃. Avoid freeze/thaw cycle. | |
Product features:
·RNase H -: Mutated HiFi MMLv reverse transcriptase with reduced RNase H activity, making it easier to obtain full-length cDNA.
·Easy to use: The reagent kit contains all the reagents required for reverse transcription, except for RNA templates.
Notes:
1. During the operation process, RNase contamination should be avoided to prevent RNA degradation or cross contamination during experiments. It is recommended to perform RNA operations in specialized areas, use specialized instruments and consumables, and have operators wear masks and disposable gloves, and frequently change gloves.
2. Disposable plastic containers should be used as much as possible for experiments. If glass containers are used, they should be treated with a 0.1% DEPC (diethyl pyrocarbonate) aqueous solution at 37 ℃ for 12 hours, and sterilized under high pressure at 120 ℃ for 30 minutes before use. Alternatively, glass containers 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. All reagents in this reagent kit should be gently mixed upside down before use, avoiding foaming as much as possible, and used after brief centrifugation. The enzymes involved should be returned to -20 ℃ as soon as possible after use to avoid repeated freeze-thaw cycles.
If the initial amount of RNA is less than 50 ng, it is recommended to add RNA enzyme inhibitors (RNAsin). This kit is not provided.
Usage:
Attention: 10 ng-5 μ G Total RNA can establish 20 μ Reaction system, if the total RNA content is greater than 5 μ g. Please expand the reaction system proportionally
i Steps for reverse transcription:
1. Dissolve RNA templates, primers, dNTP Mix, DTT, RT Buffer, HiFi MMLV, and RNase Free Water and place on ice for later use.
2. Prepare a reaction system according to the following table, with a total volume of 20 μ L.
| Reagent |
20 μlReaction system |
Final concentration |
| dNTP Mix，2.5 mM Each |
4 μl |
500 μM Each |
| Primer Mix |
2 μl |
/ |
| RNA Template |
X μl |
1 ng-5 µg |
| 5×RT Buffer |
4 μl |
1× |
| DTT，0.1 M |
2 μl |
10 mM |
| HiFi-MMLV，200 U/μl |
1 μl |
/ |
| RNase-Free Water |
up to 20 μl |
/ |
|
Attention:
1) If the initial amount of RNA is less than 50 ng, it is recommended to add RNA enzyme inhibitors (RNAsin). This kit is not provided.
2) Primer Mix is formulated from Oligo (dT) and Random Primer
3. Vortex shake and mix well, briefly centrifuge to collect the solution on the pipe wall to the bottom of the pipe.
4.
Incubate at 42 ℃ for 30-50 minutes and 85 ℃ for 5 minutes. After the reaction is complete, centrifuge briefly and cool on ice.
5. Reverse transcripts can be directly used for PCR reactions and fluorescence quantitative PCR reactions, or stored at -20 ℃ for a long time.
ii If the reverse transcription efficiency is low, or the RNA template secondary structure is complex and the GC content is high, the following steps are recommended:
1. Dissolve RNA templates, primers, dNTP Mix, DTT, RT Buffer, HiFi MMLV, and RNase Free Water and place on ice for later use.
2. Prepare the reaction system according to the following table, with a total volume of 13 μ L.
| Reagent |
20 μlReaction system |
Final concentration |
| dNTP Mix，2.5 mM Each |
4 μl |
500 μM Each |
| Primer Mix |
2 μl |
/ |
| RNA Template |
X μl |
1 ng-5 µg |
| RNase-Free Water |
up to 13 μl |
/ |
|
3.
Incubate
at 70 ℃ for 10 minutes and quickly ice bath for 2 minutes.
4. Centrifuge briefly to collect the solution on the tube wall to the bottom of the tube.
5. Continue to add the following reagents to the above reaction solution:
| Reagent |
20 μlReaction system |
Final concentration |
| 5×RT Buffer |
4 μl |
1× |
| DTT，0.1 M |
2 μl |
10 mM |
| HiFi-MMLV，200 U/μl |
1 μl |
/ |
|
Attention:
1) If the initial amount of RNA is less than 50 ng, it is recommended to add RNA enzyme inhibitors (RNAsin). This kit is not provided.
2) Primer Mix is formulated from Oligo (dT) and Random primer.
6. Gently blow and mix well, incubate at 42 ℃ for 50 minutes, and incubate at 85 ℃ for 5 minutes.
7. After the reaction is complete, centrifuge briefly and cool on ice.
8. Reverse transcripts can be directly used for PCR reactions and fluorescence quantitative PCR reactions, or stored at -20 ℃ for a long time.
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