Cas12b 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/Cas12b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。 特点是耐受65℃高温，可以与LAMP恒温扩增配合使用。
BrCas12b 是 Cas12b家族蛋白成员，来源于Brevibacillus leverages。耐高温，可以搭配LAMP恒温扩增。
组分表：
| 组分货号 | 组分名称 | 物理外观 | 100pmol | 500pmol | 2000pmol | 储存 | | B1540158A | BrCas12b (10μM) | 液体 | 10μl | 50μl | 200μl | -20℃，避免反复冻融。 | | B1540158B | Cas12b反应缓冲液 (10×) | 液体 | 200μl | 1ml | 4ml | -20℃，避免反复冻融。 | | B1540158C | Cas12b稀释缓冲液 (1×) | 液体 | 100μl | 500μl | 2ml | -20℃，避免反复冻融。 |
注：Cas12b稀释缓冲液 (1×) 即蛋白稀释液。 |
需要但未提供的试剂：
1. Reporter：Cas12b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 (Cas12 切割底物)
|
R1540212
| ssDNA-试纸型 (Cas12 切割底物) |
2. sgRNA：与BrCas12b结合，形成功能复合物，被目标序列特异性激活。推荐搭配CRISPR guide RNA转录试剂盒制备sgRNA。
(BrCas12b sgRNA scaffold sequence结构序列: 5’-GAAGGUGGUUAGCUACAGGCUGACCAGUGCAGUUGUGUCAUGUGCUACGGUGACCUAACACGUCACUCAGUCACAACGGCUAUCUAUAUUUCCACUAACCAAAGUUAGUGGAAAUGUAGAUGGUUAGCAC -3’)
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3. LAMP恒温扩增试剂盒：目标序列（DNA/ RNA）特异性扩增，提高目标序列与Cas12b蛋白酶结合效率。
检测步骤：
1 、反应体系：
| 成分名称 | 推荐用量 / 终浓度 | 优化范围 |
|---|
Cas12b反应缓冲液 (10×)
| 1× | - |
Reporter ssDNA (fluorescence）
| 250 nM
| 50 ~ 500 nM
|
sgRNA
| 250 nM
| 200 ~ 1000 nM
|
BrCas12b (10μM)
| 250 nM (=0.25μM)
| 200 ~ 500 nM (=0.2~0.5μM)
|
DNA扩增产物
| 适量 | 0-5 μL |
| ddH₂O | 补齐到 20 μL | - |
3、 结果分析：实时荧光曲线结果（示例）
.png)
阳性对照（PC：Positive control），检测样品（样本1-2）：LAMP 扩增产物激活Cas12b高效切割reporter ssDNA 释放高强度荧光信号。
阴性对照（NTC: No template control）：无模板LAMP扩增，Cas12b不切割reporter ssDNA，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 Cas12b recognizes and cleaves target DNA under the guidance of guide RNAs, its collateral cleavage activity is activated, enabling efficient non-specific cleavage of DNA in the reaction system. By designing Reporter DNA labeled with fluorophores or other markers at both ends, CRISPR/Cas12b can be used for the detection and signal amplification of DNA templates. Results can then be read via a fluorometer or lateral flow strip. It features high thermotolerance up to 65 °C and is compatible with LAMP isothermal amplification.
BrCas12b is a member of the Cas12b protein family derived from Brevibacillus sp. It is thermostable and can be coupled with LAMP isothermal amplification.
Components List:
| Component No. | Component Name | Appearance | 100pmol | 500pmol | 2000pmol | Storage | | B1540158A | BrCas12b (10μM) | Liquid | 10μl | 50μl | 200μl | -20℃. Avoid freeze/thaw cycle. | | B1540158B | Cas12b Reaction Buffer (10×) | Liquid | 200μl | 1ml | 4ml | -20℃. Avoid freeze/thaw cycle. | | B1540158C | Cas12b Diluent Buffer (1×) | Liquid | 100μl | 500μl | 2ml | -20℃. Avoid freeze/thaw cycle. |
Note：Cas12b Diluent Buffer (1×) is the protein dilution solution. |
Other materials required:
1. Reporter: Cas12b 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 (Cas12 Fluorescent Reporter)
|
R1540212
| FAM-Biotin labeled ssDNA (Cas12 Lateral Flow Reporter) |
2. sgRNA: It binds to BrCas12b to form a functional complex, which is specifically activated by target sequences. It is recommended to prepare sgRNA using the CRISPR guide RNA Transcription Kit.
(BrCas12b sgRNA scaffold sequence: 5’-GAAGGUGGUUAGCUACAGGCUGACCAGUGCAGUUGUGUCAUGUGCUACGGUGACCUAACACGUCACUCAGUCACAACGGCUAUCUAUAUUUCCACUAACCAAAGUUAGUGGAAAUGUAGAUGGUUAGCAC -3’)
3. LAMP Isothermal Amplification Kit: Specifically amplifies target sequences (DNA/RNA) to improve the binding efficiency between target sequences and BrCas12b.
Assay Protocol:
1. Reaction setup:
| Component | Recommended final concentration | Optimization range |
|---|
| Cas12b Reaction Buffer (10×) | 1× | — |
| Reporter ssDNA (fluorescence) | 250 nM | 50 ~ 500 nM |
| sgRNA | 250 nM | 200 ~ 1000 nM |
| BrCas12b (10 μM) | 250 nM (=0.25 μM) | 200 ~ 500 nM (=0.2~0.5 μM) |
| DNA amplicon | Appropriate amount | 0–5 μL |
| ddH₂O | Bring total volume to 20 μL | — |
3. Result analysis: Real-time fluorescence curve (example) .
Positive control (PC): Test samples (Sample 1–2). LAMP amplicons activate Cas12b for efficient cleavage of reporter ssDNA, producing strong fluorescence signal. Negative control (NTC: No template control): No-template LAMP reaction. Cas12b remains inactive and does not cleave reporter ssDNA, with zero fluorescence signal.
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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