RTL 逆转录酶 2.0 是一种依赖 RNA 模板的DNA 聚合酶，缺少 3' → 5' 的核酸外切酶活性， 具有 RNase H 活性。该酶可利用 RNA作为模板 ，合成一条互补的 DNA 链，可应用于第一链cDNA合成 ，特别适用于RT-LAMP（环介导等温扩增）。相较于 RTL逆转录酶 1.0，灵敏度显著提升，热稳定性更强，65℃高温反应更稳定。RTL 逆转录酶 2.0（无甘油）可用于制备冻干制剂、可冻干RT-LAMP 试剂等。
产品组分
| rp216167 | Component | 1.5 KU | Storage | | rp216167A | RTL Reverse Transcriptase (Glycerol-free) | 1.5 KU | -20°C. Avoid freeze/thaw cycle | | rp216167B | 10× HH RTL Buffer | 1.5 mL | -20°C. Avoid freeze/thaw cycle | | rp216167C | MgSO4 | 1.5 mL | -20°C. Avoid freeze/thaw cycle |
|
产品应用
1） cDNA 合成；
2） 与阿拉丁Bst DNA 聚合酶 V2 系列产品联合使用，进行 RT-LAMP 等温扩增检测靶标 RNA。
单位定义
以Poly (rA)·Oligo (dT) 为模板/引 物， 在50℃，20min 条件下，掺入1 nmol的dTTP为酸不溶性物质所需要酶量定义为 1 个活性单位 (U)。
质量控制
1、核酸内切酶残留检测：15U本酶和1μgλ-DNA，在 37℃下孵育 16h，琼脂糖凝胶电泳 DNA 谱带不发生明显变化。
2、核酸外切酶检测：15U 本酶和 1μg λ-HindIIIDNA，在 37℃下孵育 4h，琼脂糖凝胶电泳DNA 谱带不发生明显变化。
3、核酸切口酶检测：15U 本酶和 1μg pBR322DNA，在 37℃下反应 4h，琼脂糖凝胶电泳DNA 谱带缺刻<10%。
4、RNase 残留检测：15U 本酶和 0.48μg MS2 RNA 在 37℃下孵育 4h，RNA 电泳谱带完整度>90%。
5、大肠杆菌 DNA 残留检测：15U 本酶中残留的核算经 E.coli gDNA 特异性 TaqMan qPCR检测，E.coli 基因组残留<1copy。
实验流程
逆转录反应体系：
| 组分 | 体积 |
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
|
注：TotalRNA推荐使用量1ng~1μg
mRNA推荐使用量50ng~100ng
反应程序
| 温度 | 时间 |
25°Cⓐ
| 5min
|
55°C
| 10minⓑ
|
80°C
| 10min
|
ⓐ如使用RandomPrimerMix增加该孵育步骤；
ⓑ可在10-30min间调整。延长逆转录时间有助于获得更长的cDNA(>5kb)
RT-LAMP反应体系
| 组分 | 体积 | 终浓度 |
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抑制剂（40U/μL）
| 0.5 μL
| 20 U/Rxn
|
RTL逆转录酶2.0（无甘油）(15U/μL)
| 0.5 μL
| 7.5 U
|
Bst DNA聚合酶(8U/μL)
| 1 μL
| 8 U
|
MgSO4(100mM)
| 1.5 μL
| 6 mM（总共8mM）
|
10×HH RTL Buffer(或10×HH Bst V2 Buffer)*
| 2.5 μL
| 1×（含有2mM镁离子)
|
Nuclease-free Water
| Up to 25 μL
|
|
体系配制完成，涡旋混合均匀，65°C反应1h。
*注：此二者buffer互通，组分完全相同
注意事项
1）本产品在-20℃保存会形成白色固体，从 -20℃取出后放冰上 10 分钟左右，融化后震 荡混匀即可使用；
2）cDNA 产物可在-20℃或者-80℃保存或立 即用于 PCR 反应；
3）防止 RNase 污染，请保持实验区域洁净， 操作时需穿戴干净的手套、口罩。实验所用的 离心管、吸头等耗材均需保证 RNase-free。
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.
ZJ25F0319666
危险品化学品经营许可证（带存储）