RTL reverse transcriptase 2.0 is an RNA template-dependent DNA polymerase that lacks 3' → 5' exonuclease activity and has RNase H activity. The enzyme uses RNA as a template to synthesize a complementary DNA strand, which can be applied to first-strand cDNA synthesis, especially for RT-LAMP (RT-mediated isothermal amplification). Compared with RTL reverse transcriptase 1.0, the sensitivity is significantly improved, the thermal stability is stronger, and the reaction is more stable at 65℃. RTL reverse transcriptase 2.0 (glycerol free) can be used to prepare lyophilized preparations and lyophilizable RT-LAMP reagents.
Product application
1) cDNA synthesis;
2) Combined with Aladdin Bst DNA polymerase V2 series products, RT-LAMP isothermal amplification was performed to detect target RNA.
Add 4 semi-finished item numbers and packaging
rp216167A RTL reverse transcriptase 2.0 (glycerol free) (15U/μL) Package: 0.1mL
rp216167B 10 × HH RTL Buffer Package: 1.5mL
rp216167C MgSO4 (100 mM) package: 1.5mL
rp216167D Product Manual Package: 1EA
Unit definition
Using Poly (rA)·Oligo (dT) as template/primer, the amount of enzyme required to incorporate 1 nmol of dTTP as acid-insoluble substance was defined as 1 activity unit (U) at 50℃ for 20min.
Quality control
1, endonucrenase residue detection: 15U benzyme and 1μgλ-DNA, incubated at 37℃ for 16h, agarose gel electrophoresis DNA band did not change significantly.
2. Exonuclidenase detection: 15U benzyme and 1μg λ-HindIIIDNA were incubated at 37℃ for 4h, and the DNA bands of agarose gel electrophoresis did not change significantly.
3, nucleic acid incision enzyme detection: 15U benzyme and 1μg pBR322DNA, reaction at 37℃ for 4h, agarose gel electrophoresis DNA band notch <10%.
4. RNase residue detection: 15U this enzyme and 0.48μg MS2 RNA were incubated at 37℃ for 4h, and the band integrity of RNA electrophoresis was >90%.
5. Detection of Escherichia coli DNA residue: Accounting of residues in this enzyme by E.coli GDNA-specific TaqMan qPCR detection, E.coli genome residue <1copy.
Experimental process
Retrotranscriptional response system:
Constituent
| 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 × HH RTL Buffer
| 2 μL
|
Nuclease-free Water
| Up to 20 μL |
Note: The recommended amount of TotalRNA is 1ng~1μg
The recommended dosage of mRNA is 50ng~100ng
Reaction program
Temperature
| Time |
25°Cⓐ
| 5min
|
55°C
| 10minⓑ
|
80°C
| 10min
|
ⓐIf RandomPrimerMix is used to increase the incubation step;
ⓑIt can be adjusted between 10 and 30 minutes. Longer reverse transcription time helps to obtain longer cDNA(>5kb)
RT-LAMP reaction system
Constituent
| Volume
| Final concentration
|
Template RNA
| optional
| ≥10copies
|
dNTPMix(10mM)
| 3.5 μL
| 1.4 mM
|
FIP/BIPPrimers(25×)
| 1 μL
| 1.6 μM
|
F3/B3Primers(25×)
| 1 μL
| 0.2 μM
|
LoopF/LoopBPrimers(25×)
| 1 μL
| 0.4 μM
|
RNase inhibitor（40U/μL）
| 0.5 μL
| 20 U/Rxn
|
RTL Reverse Transcriptase (Glycerol-free) (15U/μL)
| 0.5 μL
| 7.5 U
|
Bst DNA polymerase (8U/μL)
| 1 μL
| 8 U
|
MgSO4 (100mM)
| 1.5 μL
| 6 mM（Total 8mM）
|
10×HHRTLBuffer(or 10×HHBstV2Buffer)*
| 2.5 μL
| 1×（with 2mM Mg)
|
Nuclease-free Water
| Up to 25 μL
|
|
The system was prepared, the vortex was mixed evenly, and the reaction was 1h at 65°C.
* Note: The two buffer interwork, the composition is exactly the same
Matters needing attention
1) The product will form a white solid when stored at -20℃. Put it on ice for about 10 minutes after taking it out at -20℃. After melting, shake and mix it well.
2) The cDNA product can be stored at -20℃ or -80℃ or immediately used for PCR reaction;
3) To prevent RNase contamination, please keep the experiment area clean and wear clean gloves and masks during operation. The centrifugal tube, suction and other consumables used in the experiment should be guaranteed to be RNase-free.
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