| Product Description | HiFi II M-MLV (H -) is a reverse transcriptase that recombines and expresses mutated M-MLV genes using E. coli engineering bacteria. This enzyme can catalyze complementary DNA polymerization reactions using RNA or DNA: RNA hybrid strands as templates. The mutated HiFi II M-MLV (H -) reverse transcriptase RNase H activity is missing, reducing RNA degradation in reverse transcription reactions and making it easier to obtain full-length cDNA. HiFi II M-MLV (H -) reverse transcriptase can synthesize the first strand of cDNA at 55 ℃, providing higher specificity, strong stability, and can synthesize up to 12 kb of cDNA with high cDNA yield. Suitable for the synthesis of first stranded cDNA, RT PCR, RT qPCR, and construction of full-length cDNA libraries. | H665664 | Component | 10 KU | Storage | | H665664A | HiFi II M-MLV(H-) (200 U/μL) | 50 μL | -20℃. Avoid freeze/thaw cycle. | | H665664B | 5×SuperRT Buffer | 1 mL | -20℃. Avoid freeze/thaw cycle. |
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Activity definition:
Using Poly (A) as a template and oligo (dT) as a primer, the enzyme required to catalyze the addition of 1 nmol of dTTP within 10 minutes at 37 ℃ is defined as one active unit (U).
Quality control:
200 U of this enzyme reacted with 1 µ g of 16 S, 23 S rRNA at 37 ℃ for 1 hour, and the electrophoresis band of the RNA remained unchanged.
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 L reaction system, if the total RNA amount is greater than 5 μ g. Please expand the reaction system proportionally.
i Steps for reverse transcription:
1. Dissolve the RNA template, primers, dNTP Mix, SuperRT Buffer, HiFi II M-MLV (H -), and RNase Free Water and place them 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 |
Oligo-dT Primer，100 µ M
Or Random Primers ，50 μ M
or Specific Primer , 10 μ M |
1 μl |
/ |
| RNA Template |
X μl |
1 ng-5 µg |
| 5×SuperRT Buffer |
4 μl |
1 × |
| HiFi II M-MLV(H-) (200U /μL) |
0.5-1 μL |
/ |
| RNase-Free Water |
up to 20 μL |
/ |
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Note: If the initial amount of RNA is less than 50ng, it is recommended to add RNA enzyme inhibitors (RNasins). This kit is not provided.
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 55 ℃ for 1-30 minutes, and incubate at 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 the RNA template, primers, dNTP Mix, SuperRT Buffer, HiFi II M-MLV (H -), and RNase Free Water and place them on ice for later use.
2. Prepare a reaction system according to the following table, with a total volume of 15 μ L.
| Reagent |
20 μlReaction system |
Final concentration |
| dNTP Mix，2.5 mM Each |
4 μl |
500 μM Each |
Oligo-dT Primer，100 µ M
Or Random Primers ，50 μ M
or Specific Primer , 10 μ M |
1 μl |
/ |
| RNA Template |
X μl |
1 ng-5 µg |
| RNase-Free Water |
up to 15 μL |
/ |
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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. Add 4 to the above reaction solution μ L 5 x SuperRT Buffer.
Note: If the initial amount of RNA is less than 50 ng, it is recommended to add RNA enzyme inhibitors (RNasins). This kit is not provided.
6. Gently blow and mix well. If the reverse transcription primer is Oligo dT Primer or Specific Primer,
7. Incubate at 42 ℃ for 2 minutes; If the reverse transcription primer is Random Primers, incubate at 25 ℃ for 10 minutes.
8. Join 1 μ L HiFi II M-MLV (H -) (200 U/ μ L) Gently pat and mix well. Incubate at 55 ℃ for 50 minutes. Incubate at 85 ℃ for 5 minutes. After the reaction is complete, centrifuge briefly and cool on ice.
9. 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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