RTL Reverse Transcriptase
RTL reverse transcriptase is an RNA template-dependent DNA polymerase that lacks the 3'→5' exonuclease activity and has RNase H activity. This enzyme can use RNA as a template to synthesize a complementary strand of DNA, 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 sensitivity is significantly improved, the thermal stability is stronger, and the reaction at 65°C is more stable. RTL reverse transcriptase (glycerol free) can be used to prepare lyophilized preparations, lyophilized RT-LAMP reagents etc.
Unit Definition
One unit incorporates 1 nmol of dTTP into acid-precipitable material in 20 minutes at 50°C using poly(A)•oligo(dT)25 as template-primer.
Components
|
Component |
HC5008A-01 |
HC5008A-02 |
HC5008A-03 |
|
RTL Reverse Transcriptase (Glycerol-free) (15U/μL) |
0.1 mL |
1 mL |
10 mL |
|
10×HC RTL Buffer |
1.5 mL |
4×1.5 mL |
5×10 mL |
|
MgSO4 (100mM) |
1.5 mL |
2×1.5 mL |
3×10 mL |
Storage Condition
Transportation under 0°C and be stored at -25°C~-15°C.
Quality Control
- Residual Activity of Endonuclease: A 50 μL reaction containing 1 μg of λDNA and 15 units of RTL2.0 incubated for 16 hours at 37 ℃ shows same pattern as negative control by gel electrophoresis.
- Residual Activity of Exonuclease: A 50 μL reaction containing 1 μg of Hind Ⅲ digested λDNA and 15 units of RTL2.0 incubated for 16 hours at 37 ℃ shows same pattern as negative control by gel electrophoresis.
- Residual Activity of Nickase: A 50 μL reaction containing 1 μg of supercoiled pBR322 and 15 units of RTL2.0 incubated for 4 hours at 37°C shows same pattern as negative control by gel electrophoresis.
- Residual Activity of RNase: A 10 μL reaction containing 0.48 μg of MS2 RNA and 15 units of RTL2.0 incubated for 4 hours at 37°C shows same pattern as negative control by gel electrophoresis.
- E. coli gDNA: Measured with E.coli specific HCD detection kits，15 units of RTL2.0 contains less than 1 E. coli genome.
Reaction Setup
cDNA Synthesis Protocol
|
Components |
Volume |
|
Template RNA a |
optional |
|
Oligo(dT) 18~25(50uM) or Random Primer mix(60uM) |
2 μL |
|
dNTP Mix (10mM each) |
1 μL |
|
RNase Inhibitor (40U/uL) |
0.5 μL |
|
RTL Reverse Transcriptase 2.0 (15U/uL) |
0.5 μL |
|
10×HC 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
Thermo-cycling Conditions for a routine reaction：
|
Temperature (°C) |
Time |
|
25 °C a |
5mins |
|
55 °C |
10mins b |
|
80 °C |
10mins |
Notes:
1) If Random Primer Mix is used,an incubation step at 25°C.
2) If target primer mix is used, an incubation step at 55°C for 10~30mins.
RT-LAMP Protocol
|
Components |
Volume |
Final Concentration |
|
Template RNA |
optional |
≥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/Reaction |
|
RTL Reverse Transcriptase 2.0 (15U/μL) |
0.5 μL |
7.5 U/Reaction |
|
Bst V2 DNA Polymerase (8U/μL) |
1 μL |
8 U/Reaction |
|
MgSO4 (100mM) |
1.5 μL |
6 mM (Total 8 mM) |
|
10×HC RTL Buffer (or 10×HC Bst V2 Buffer) |
2.5 μL |
1 × (2mM Mg2+) |
|
Nuclease-free Water |
Up to 25 μL |
- |
Notes:
1) Mix by vortexing and centrifuge briefly to collect. Constant temperature incubation at 65°C for 1 hour.
2) The two buffers are interoperable and have the same composition.
Notes
1.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.
2.The cDNA product could be stored at -20°C or -80°C or used immediately for PCR reaction.
3.To prevent RNase contamination, please keep the experimental area clean, and wear clean gloves and masks during operation.
