RTL Reverse Transcriptase（Glycerol free, high density）
RTL reverse transcriptase 2.0 is an RNA template-dependent DNA polymerase that lacks 3′ →5′ exonuclease activity and has RNaseH activity. The enzyme can use RNA as a template to synthesize a complementary DNA strand, which can be applied to first-strand cDNA synthesis, especially for RT-LAMP (Loop-mediated isothermal amplification). Compared with RTL reverse transcriptase 1.0, the enzyme is significantly more sensitive and thermally stable. RTL reverse transcriptase 2.0 (high concentration glycerol free) can be used to prepare lyophilized preparations, lyophilizable RT-LAMP reagents, etc.
Components
|
Composition |
1500 U |
15000 U |
15000 U |
|
RTL Reverse Transcriptase 2.0 (High concentration glycerol-free)（75U/μL） |
0.02mL |
0.2 mL |
2 mL |
|
10 × RTL Buffer |
1.5mL |
4×1.5mL |
5×10mL |
|
MgSO4 (100mM) |
1.5mL |
2×1.5mL |
3×10mL |
Storage Condition
Transported under 0°C and be stored at -25°C~ -15°C. Avoid repeated freezing and thawing, and valid for 18 months.
Unit definition
One unit incorporates 1 nmol of dTTP into acid-precipitable material in 20 minutes at 50°C using poly(A)•oligo(dT) as template-primer.
Quality Control
Endonuclease Activity: Incubation of 15 U of enzyme with 1 μg λ DNA for 16 hours at 37°C resulted in no detectable degradation of the DNA as determined by gel electrophoresis.
•Exonuclease Activity: Incubation of 15U of enzyme with 1 μg λ-Hind III digest DNA for 4 hours at 37°C resulted in no detectable degradation of the DNA as determined by gel
electrophoresis.
•Nickase Activity: Incubation of 15 U of enzyme with 1μg pBR322 for 4 hours at 37°C resulted in no detectable degradation of the DNA as determined by gel electrophoresis.
•RNase Activity: Incubation of 15 U of enzyme with 0.48μg MS2 RNA for 4 hours at 37 ℃ resulted in no detectable degradation of the RNA as determined by gel electrophoresis.
•E.coli DNA: 15 U of enzyme is detected by TaqMan qPCR. The E.coli DNA is <1 copy.
Reaction Setup
cDNA Synthesis Protocol
|
Components |
Volume |
|
Template RNA a |
Variable |
|
Oligo(dT) 18~25(50μM) or Random Primer mix(60μM) |
2 μL |
|
dNTP Mix (10mM each) |
1 μL |
|
RNase Inhibitor (40U/μL) |
0.5 μL |
|
RTL Reverse Transcriptase 2.0 (High concentration glycerol-free)（75U/μL） |
0.1 μL |
|
10× RTL Buffer |
2 μL |
|
Nuclease-free Water |
Up to 20 μL |
Notes:
1) The recommended dosage of Total RNA is 1ng~1μg
2) The recommended dosage of mRNA was 50ng~100ng
Thermocycling conditions for a routine reaction：
|
Temperature (°C) |
Time |
|
25 °C a |
5mins |
|
55 °C |
10mins b |
|
80 °C |
10mins |
Notes:
1) a .If Random Primer Mix is used, an incubation step at 25°C.
2) b. If target primer mix is used, an incubation step at 55℃for 10~30mins. Longer reverse transcription time helps to obtain longer cDNA (>5 kb).
RT-LAMP Protocol
|
Components |
Volume |
Final Concentration |
|
Template RNA |
Variable |
≥10 copies |
|
dNTP Mix (10mM) |
3.5 μL |
1.4 mM |
|
FIP/BIP Primers (25×) |
1 μL |
1.6 μM |
|
F3/B3 Primers (25×) |
1 μL |
0.2 μM |
|
LoopF/LoopB Primers (25×) |
1 μL |
0.4 μM |
|
RNase Inhibitor (40U/μL) |
0.5 μL |
20 U |
|
RTL Reverse Transcriptase 2.0 (High concentration glycerol-free) (75 U/μL) |
0.1 μL |
7.5 U |
|
Bst DNA Polymerase (8U/μL) |
1 μL |
8 U |
|
MgSO4 (100mM) |
1.5 μL |
6 mM (Total 8 mM) |
|
10×RTL Buffer (or 10×Bst V2 Buffer) |
2.5 μL |
1 × |
|
Nuclease-free Water |
Up to 25 μL |
- |
Notes:
Mix by vortexing and centrifuge briefly. Then keep at 65°C for 1 hour.
The two buffers are interoperable and have the same composition.
Notes
- This product will form a white solid when stored at -20 °C. Take it out from -20°C and put it on ice for about 10 minutes. After melting, it can be used by shaking and mixing.
- The cDNA product could be stored at -20°C or -80°C or used immediately for PCR reaction.
- To prevent RNase contamination, please keep the working area clean, and wear cleaned gloves and masks during operation.The centrifugal tube, suction and other consumables used in the experiment should be guaranteed to be RNase-free.
