Cat.No. |
14608ES92 / 14608ES97 / 14608ES03 |
Size |
10,000 U / 50,000 U/ 1 mL |
Unit Definition |
One unit is defined as the amount of enzyme that will incorporate 1 nmol of dTTP into acid-insoluble material in 10 minutes at 37°C using Oligo(dT) as primers. |
Components No. |
Name |
14608ES92 |
14608ES97 |
14608ES03 |
14608-A |
Hifair UCF.METM V Reverse Transcriptase (200 U/μL) |
50 μL |
250 μL |
1 mL |
14608-B |
5×HifairTM V Buffer |
250 μL |
1.25 mL |
5 mL |
This product should be stored at -25~-15℃ for 2 years.
1. Denaturation of RNA template (This step is optional, denaturation of RNA template helps to open the secondary structures, which will improve the yield of the first strand cDNA.)
Components |
Volume (μL) |
RNase free ddH2O |
to 13 |
Oligo (dT)18 (50 μmol/L) or Random Primers (50 μmol/L) or Gene Specific Primers (2 μmol/L) |
1 |
or 1 |
|
or 1 |
|
RNA template |
X * |
*Total RNA: 1~5 μg or mRNA: 1~500 ng
Incubating at 65°C for 5 minutes, then transferring on ice immediately to chill for 2 minutes. Brief centrifugation to collect reaction liquid, add the reverse transcription reaction solution as shown in the following table. Gently pipette to mix.
2. Preparation of the reaction mixture (20 μL volume)
Components |
Volume (μL) |
Mixture of previous step |
13 |
5×HifairTM V Buffer |
4 |
dNTP Mix (10 mmol/L) |
1 |
Hifair UCF.METM V Reverse Transcriptase (200 U/μL) |
1 |
RNase inhibitor (40 U/µL) |
1 |
3. Perform the reaction under the following conditions
Temperature |
Time |
25°C* |
5 min |
42°C** |
15-30 min |
85°C*** |
5 min |
* Incubating at 25°C for 5 min is required only for using the random hexamers. Please skip this step when using Oligo (dT)18 or Gene Specific Primer.
**The recommended reverse transcription temperature is 42°C. For templates with complicated secondary structures or high GC content, it is recommended to raise the reaction temperature to 50~55°C.
***Heating at 85°C for 5 min to inactivate reverse transcriptase.
The product can be directly used in PCR or qPCR reactions, or stored at -20°C for short-term storage. It is recommended to aliquot the products and store at -80°C for long-term storage. Avoid repeated freezing and thawing.
The product is suitable for one-step RT-qPCR, it is recommended to add 10~20 U reverse transcriptase for every 25 μL reaction system, or gradually increase the amount of reverse transcriptase according to the actual situation.
1. Please keep the experiment area clean; Wear clean gloves and masks when operating. The consumables used in the experiment should be guaranteed to be RNase free to prevent RNase contamination.
2. All operations should be performed on ice to prevent RNA degradation.
3. To ensure efficient reverse transcription, it is recommended to use high quality RNA samples.
4. This product is for research use only.
5. Please operate with lab coats and disposable gloves, for your safety.
Ver.EN20230918
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