Cas12 在向导 RNA 引导下识别并切割靶标 DNA时， 其“附属切割” 活性被激活， 可高效切 割体系中非特异DNA。 通过设计两端标记荧光基团或者其他标记物的Reporter DNA ， 可实现 CRISPR/Cas12 对 DNA 模板的检测和信号放大。然后通过荧光仪或试纸观察结果。
LbaCas12a (Cpf1) 是 Cas12a 家族蛋白成员，来源于 Lachnospiraceae。与crRNA结合，特异性识别DNA序列，反式切割ssDNA。
组分表：
| 组分货号 | 组分名称 | 物理外观 | 100pmol | 500pmol | 2000pmol | 储存 | | L1540156A | LbaCas12a (10μM) | 液体 | 10μl | 50μl | 200μl | -20℃，避免反复冻融。 | | L1540156B | Cas12a反应缓冲液 (10×) | 液体 | 200μl | 1ml | 4ml | -20℃，避免反复冻融。 | | L1540156C | Cas12a稀释缓冲液 (1×) | 液体 | 100μl | 500μl | 2ml | -20℃，避免反复冻融。 |
注：
1. LbaCas12a 采用含 50% 甘油的储存液保存。
2. Cas12a稀释缓冲液 (1×) 为 Cas12a 蛋白稀释液，含 50% 甘油。
如用于冻干应用，可提供无甘油版本定制服务。 |
活性温度范围： 25℃~55℃。
需要但未提供的试剂：
1. Reporter：Cas12a切割底物。可搭配我司的Reporter进行检测，或自行设计合成Reporter。
| 货号 | 名称 |
|---|
R1540211
| ssDNA-荧光型 (Cas12a 切割底物)
|
R1540212
| ssDNA-试纸型 (Cas12a 切割底物) |
2. crRNA/sgRNA：与Cas12a结合，形成功能复合物，被目标序列特异性激活。
(LbaCas12a crRNA scaffold sequence结构序列: 5’-UAAUUUCUACUAAGUGUAGAU-3’)
推荐搭配CRISPR guide RNA转录试剂盒制备sgRNA。
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3. 目标DNA。推荐搭配RPA/RAA恒温扩增试剂盒。
检测步骤：
1 、目标序列（DNA/RNA）特异性扩增。可以采用PCR，RPA，RAA，LAMP，RCA等扩增技术。
2 、根据下表配置反应体系：
| 成分名称 | 推荐用量 / 终浓度 | 优化范围 |
|---|
Cas12a反应缓冲液 (10×)
| 1× | - | | Reporter (FAM-BHQ1 labelled ssDNA) | 120 nM | 40 ~ 200 nM | | crRNA | 50 nM | 20 ~ 200 nM | LbaCas12a (10μM)
| 20 nM* (=0.02 μM) | 10 ~ 100 nM (=0.01 μM~0.1 μM) | | 上步反应的扩增产物 | 适量 | 0-5 μL | | H₂O | 补齐到 20 μL | - |
* 1、每家公司的酶活性不同，反应缓冲体系不同，所以酶的使用量不同。每个项目的Cas12a 最佳使用浓度也不同。初次使用，建议拉梯度浓度对比测试。例如：Cas12a终浓度依次为100nM, 50nM, 25nM, 12.5nM 进行横向对比。
2、 Cas蛋白母液浓度为10 μM。建议使用稀释缓冲液将其按1:20比例稀释至0.5 μM的工作 浓度（例如，取1 μL蛋白母液与19 μL 稀释缓冲液混合）。随后，取0.8 μL稀释后的蛋白溶液，加入至20 μL的反应体系中，此时Cas蛋白的终浓度为20nM。
3、建议在反应前将Cas蛋白与crRNA预混，使二者预先形成稳定的效应复合物，这不仅能避免后续组分竞争性结合，还能有效提高酶活稳定性及复杂样本中的检测耐受性。
|
3、设置反应温度 37℃， 每 1 分钟读取一次荧光数值， 反应时间 20~60分钟。
4、结果分析：实时荧光仪读数（示例）
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Positive control：在含有target template 的情况下，Cas12a高效切割reporter DNA 释放高 强度荧光信号。
Negative control：无target template的情况，Cas12a不切割reporter DNA，荧光信号为 0。
注意事项：
1. 本产品仅供科研使用， 使用前请仔细阅读本说明书。
2. 请严格按照基因扩增实验室的管理规范进行实验操作， 注意防止扩增产物污染。
3. 实验结束后， 检测过程中所产生的废弃物应按照相关规范进行处理。
When Cas12 recognizes and cleaves target DNA under the guidance of guide RNA, its collateral cleavage activity is activated, enabling efficient cleavage of non‑specific DNA in the reaction system. By designing reporter DNA labeled with fluorophores or other tags at both ends, CRISPR/Cas12 can be used for the detection and signal amplification of DNA templates. Results can then be read via a fluorescence detector or lateral flow strip.
LbaCas12a (Cpf1) is a member of the Cas12a protein family, derived from Lachnospiraceae. It binds to crRNA, specifically recognizes DNA sequences, and exhibits trans‑cleavage activity toward ssDNA.
Components List:
| Component No. | Component Name | Appearance | 100pmol | 500pmol | 2000pmol | Storage | | L1540156A | LbaCas12a (10μM) | Liquid | 10μl | 50μl | 200μl | -20℃. Avoid freeze/thaw cycle. | | L1540156B | Cas12a Reaction Buffer (10×) | Liquid | 200μl | 1ml | 4ml | -20℃. Avoid freeze/thaw cycle. | | L1540156C | Cas12a Diluent Buffer (1×) | Liquid | 100μl | 500μl | 2ml | -20℃. Avoid freeze/thaw cycle. |
Note: 1. LbaCas12a is provided in storage buffer with 50% glycerol. 2. Cas12a Diluent Buffer (1×) is for protein dilution and contains 50% glycerol. Glycerol‑free formulation is available upon request for lyophilization. |
Active temperature range: 25 °C ~ 55 °C.
Other materials required:
1. Reporter: Cas12a cleavage substrate. Detection can be performed using our reporter, or custom‑designed reporter can be synthesized independently.
Cat. No.
| Item |
|---|
R1540211
| FAM-BHQ1 labeled ssDNA (Cas12a Fluorescent Reporter)
|
R1540212
| FAM-Biotin labeled ssDNA (Cas12a Lateral Flow Reporter) |
2. crRNA/sgRNA: Binds to Cas12a to form a functional complex, which is specifically activated by the target sequence.
(LbaCas12a crRNA scaffold sequence: 5′-UAAAUUUCUACAAAGUGUAGAU-3′)
It is recommended to prepare sgRNA with the CRISPR guide RNA transcription kit.
3. Target DNA. RPA/RAA isothermal amplification kit is recommended for pairing.
Assay Protocol:
1. Specific amplification of target sequence (DNA/RNA). Amplification methods including PCR, RPA, RAA, LAMP and RCA are available.
2. Prepare the reaction system according to the table below:
| Component | Recommended dosage / final concentration | Optimization range |
|---|
| Cas12a Reaction buffer (10×) | 1× | - | | Reporter (FAM-BHQ1 labelled ssDNA) | 120 nM | 40 ~ 200 nM | | crRNA | 50 nM | 20 ~ 200 nM | LbaCas12a (10μM)
| 20 nM* (=0.02 μM) | 10 ~ 100 nM (=0.01 μM~0.1 μM) | | Amplification product from the previous step | Appropriate amount | 0-5 μL | | H₂O | Bring up to 20 μL | - |
Notes: * 1. Enzyme dosage varies among manufacturers due to differences in enzyme activity and buffer formulation. The optimal Cas12a concentration is assay-dependent. A gradient test is recommended for first-time use. For example, test Cas12a final concentrations of 100 nM, 50 nM, 25 nM and 12.5 nM side by side. 2. The stock concentration of Cas protein is 10 μM. Dilute it 1:20 with diluent buffer to 0.5 μM working concentration (e.g., mix 1 μL protein stock with 19 μL diluent buffer). Then add 0.8 μL of the diluted protein into the 20 μL reaction system for a final Cas concentration of 20 nM. 3. Pre-incubate Cas protein with crRNA prior to reaction to form stable effector complexes. This avoids competitive binding with subsequent components and improves enzyme stability and assay tolerance in complex samples. |
3. Incubate the reaction at 37 °C. Read fluorescence signals every 1 min for 20–60 min.
4. Result analysis: Real-time fluorescence readings (example).
Positive control: In the presence of the target template, Cas12a efficiently cleaves the reporter DNA and generates a strong fluorescence signal.
Negative control: In the absence of the target template, Cas12a does not cleave the reporter DNA, and the fluorescence signal remains at baseline.
Precautions
1. For research use only. Please read this manual carefully before use.
2. Operate in strict accordance with the management specifications for gene amplification laboratories to prevent amplicon contamination.
3. Waste generated during the assay shall be disposed of in accordance with relevant regulations after the experiment.
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