RTL Reverse Transcriptase（Glycerol free） HYB212
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. RTL reverse transcriptase (glycerol free) can be used to can be used to prepare freeze-dried preparations, freeze-dried RT-LAMP reagents, etc.
Components
|
Components |
1500U |
15000U |
150000U |
|
RTL Reverse Transcriptase (Glycerol-free) (15U/μL) |
0.1mL |
1mL |
10mL |
|
10 × HH RTL Buffer |
1.5mL |
4×1.5mL |
5×10mL |
|
MgSO4（100 mM） |
1.5 mL |
2×1.5 mL |
3×10 mL |
Storage
Store at -25～-15 °C, 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)25 as template-primer.
Quality control
1. Residual Activity of Endonuclease: A 50µL reaction containing 1 µg of λDNA and 15 units of RTL incubated for 16 hours at 37ºC shows same pattern as negative control by gel electrophoresis.
2. Residual Activity of Exonuclease: A 50µL reaction containing 1 µg of Hind III digested λDNA and 15 units of RTL incubated for 4 hours at 37°C shows same pattern as negative control by gel electrophoresis.
3. Residual Activity of Nickase: A 50 µL reaction containing1 µg of supercoiled pBR322 and 15 units of RTL incubated for 4 hours at 37ºC shows same pattern as negative control by gel electrophoresis.
4. Residual Activity of RNase:A 10 µL reaction containing 0.48 µg of MS2 RNA and 15 units of RTL incubated for 4 hours at 37ºC shows same pattern as negative control by gel electrophoresis.
5. E.coligDNA: Measured with E.coli specific HCD detection kits，15 units of RTL contains less than 1 E. coli genome.
Reverse transcription reaction system
|
Component |
Volume |
|
Template RNA |
optional |
|
Oligo(dT)18~25（50μM）or Random Primer Mix（60 μM） |
2 μL |
|
dNTP mix (10 mM each) |
1 μL |
|
RNase Inhibitor（40 U/μL） |
0.5 μL |
|
RTL Reverse Transcriptase (Glycerol-free) (15U/μL) |
0.5 μL |
|
10 x HH RTL Buffer |
2 μL |
|
Nuclease-free Water |
Up to 20 μL |
Note: The recommended dosage of Total RNA is 1ng~1μg. The recommended dosage of mRNA was 50ng~100ng.
Reaction program
|
Temperature |
Time |
|
25 ºC* |
5 min |
|
55 ºC |
10 min** |
|
80 ºC |
10 min |
*If Random Primer Mix is used，an incubation step at 25°C.
**If target primer mix is used, an incubation step at 55°C for 10~30 min.
RT-LAMP reaction system
|
Component |
Volume |
Final concentration |
|
Template RNA* |
optional |
≥10copies |
|
dNTP Mix (10 mM) |
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（40 U/μL） |
0.5 μL |
20U/Rxn |
|
RTL Reverse Transcriptase (Glycerol-free) (15U/μL) |
0.5 μL |
7.5 U |
|
Bst 2.0 DNA Polymerase (8 U/μL) |
1 μL |
8 U |
|
MgSO4 (100 mM) |
1.5 μL |
6 mM (Total 8mM) |
|
10 x HH RTL Buffer / 10 x HH Bst 2.0 Buffer |
2.5 μL |
1 ×(2 mM Mg2+) |
|
Nuclease-free Water |
Up to 25 μL |
- |
Mix by vertexing and centrifuge briefly to collect. Constant temperature incubation at 60°C-65°C for 1 hour.
Notes
1. This product will form a white solid whenstored 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.
3. To prevent RNase contamination, please keep the experimental area clean, and wear clean gloves and masks during operation.