M-MLV Reverse Transcriptase
RevScript Reverse transcriptase is obtained by genetic engineering technology. It has higher cDNA synthesis ability, thermal stability and reaction temperature limit (up to 60°C). The synthesized cDNA product is up to 10 kb. It enhances the affinity of the templates and is suitable for reverse transcription of RNA templates with complex secondary structure or low copy genes.
Components
|
Component |
HC2003B-01 (10,000U) |
HC2003B-02 (5*10,000U) |
HC2003B-03 (200,000U) |
|
RevScript Reverse Transcriptase (200U/μL) |
50 μL |
5×50 μL |
1 mL |
|
5 × RevScript Buffer |
250 μL |
1.25 mL |
5 mL |
Storage Condition
This product should be stored at -25°C~-15°C for 2 years.
Unit Definition
One unit incorporates 1 nmol of dTTP into acid-insoluble material in 10 minutes at 37°C using Oligo(dT) as primers.
Reaction Setup
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 Primer (50 μmol/L) Or Gene Specific Primers (2 μmol/L) |
1 |
|
or 1 |
|
|
or 1 |
|
|
RNA template |
X a |
Notes:
1) a: Total RNA: 1-5 ug or mRNA: 1-500 ng
2) 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.
1.Preparation of the reaction mixture (20 μL volume)
|
Components |
Volume (μL) |
|
Mixture of previous step |
13 |
|
5×Buffer |
4 |
|
dNTP Mix (10nmol/L) |
1 |
|
Reverse Transcriptase (200 U/μL) |
1 |
|
RNase inhibitor (40 U/μL) |
1 |
1.Perform the reaction under the following conditions:
|
Temperature (°C) |
Time |
|
25 °C a |
5mins |
|
42 °C b |
15-30mins |
|
85 °C c |
5mins |
Notes:
1) a. Incubating at 25°C for 5mins is required only for using the random hexamers. Please skip this step when using Oligo (dT)18 or Gene Specific Primer.
2) b. 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.
3) c. Heating at 85°C for 5mins to inactivate reverse transcriptase.
4) The product can be directly used in PCR or qPCR reactions, or stored at -20°C for short-term storage. It is recommended to aliguot the products and store at -80°C for long-term storage. Avoid frequent freeze-thaw.
5) The product is suitable for one-step RT-qPCR, it is recommended to add 10-20 U reverse transcriptase for every 25μL reaction system, or gradually increase the amount of reverse transcriptase according to the actual situation.
Notes
1.Please keep the experimental area clean; Clean gloves and masks should be worn during operation. All the consumables used in the experiment should be RNase free to prevent RNase contamination.
2.All procedures should be performed on ice to prevent RNA degradation.
3.High quality RNA samples are recommended to ensure high efficiency of reverse transcription.
4.This product is for research use only.
5.Please operate with lab coats and disposable gloves, for your safety.
