阿拉丁快速内切酶BamHI是一种经过基因工程重组的快速限制性内切酶，适用于质粒DNA、PCR产物或基因组DNA等的快速酶切。该酶在通用的10×缓冲液或10×彩色缓冲液中均具有优良的活性，能够在5～15分钟内完成酶切。
10×彩色缓冲液包括红色和黄色示踪染料，可将产物直接用于凝胶电泳。红色染料与2500 bp双链DNA片段在1%琼脂糖凝胶中迁移速率接近；黄色染料与10 bp双链DNA片段在1%琼脂糖凝胶中迁移速率接近。
组分与储存
各组件产品号 | 组件名称 | 物理外观 | 500 rxns | 储存温度 |
L358964-A | BamHI | 液体 | 500 μL | -20 ℃ |
L358964-B | 10×缓冲液 | 液体 | 2×1 mL | -20 ℃ |
L358964-C | 10×彩色缓冲液 | 液体 | 2×1 mL | -20 ℃ |
建议反应条件
1×缓冲液；
37℃温育；
参照“DNA快速酶切流程”配制反应体系。
失活条件
不可热失活，请使用酚氯仿抽提或柱纯化。
质量控制
1、功能活性检测
37℃下，在20 μL通用缓冲液反应体系中，1 μL快速内切酶BamHI能够在15 min内完全消化1 μg λDNA。
2、超长时间温育检测
37℃下，在20 μL通用缓冲液反应体系中，将1 μL快速内切酶BamHI与1 μg λDNA共同温育3 h，未检测到其他核酸酶污染或星号活性引起的底物非特异性降解。更长时间酶切可能出现星号活性。
3、酶切-连接-再酶切检测
37℃下，使用10倍酶量的快速内切酶BamHI消化DNA底物，回收酶切产物，在22℃下使用T4 DNA Ligase (Fast)可以将超过95%的酶切产物重新连接。将连接产物再次回收后，使用相同的内切酶可以重新切开95%以上的连接产物。
4、非特异性内切酶活性检测
37℃下，在20 μL通用缓冲液反应体系中将1 μL快速内切酶BamHI与1 μg超螺旋质粒DNA共同温育4 h后，使用琼脂糖凝胶电泳检测，少于10%的质粒DNA转变成缺刻或线性状态。
5、蓝白斑检测
使用1 μL快速内切酶BamHI消化含有lacZα基因且仅在该基因上具有1个酶切位点的特定载体。将酶切产物重新连接后转化到大肠杆菌感受态细胞中，涂布在含有X-gal、IPTG和相应抗生素的LB平板培养基上生长。成功连接的β-半乳糖苷酶基因可以正确表达，并生长出蓝色菌落；而因酶切末端降解等原因未能重新连接的产物将得到白色菌落。对于快速内切酶BamHI而言，白色菌落的比例应当小于1%。
使用方法
1. DNA快速酶切流程
① 在冰上按如下建议的加样顺序配制反应体系：
组分 | 质粒DNA | PCR产物 | 基因组DNA |
ddH₂O | 15 μL | 16 μL | 30 μL |
10×缓冲液或10×彩色缓冲液 | 2 μL | 3 μLᵃ | 5 μL |
底物DNA | 2 μL (最多1 μg) | 10 μL (约0.2 μg) | 10 μL (5 μg) |
BamHI | 1 μL | 1 μL | 5 μL |
总体积 | 20 μL | 30 μL | 50 μL |
注a：本体系适用于经过纯化的PCR产物酶切。未纯化的PCR产物具备一定的离子强度，10×缓冲液加入量可适当减少至2 μL。但由于DNA聚合酶同时具有外切酶活性，会影响酶切产物，因此如下一步需进行克隆等操作，建议酶切前对PCR产物进行纯化。
② 轻柔吸打或轻弹管壁以混匀（切勿涡旋），然后瞬时离心以收集挂壁液滴。
③ 37℃温育15 min（质粒），或15～30 min（PCR产物），或30～60 min（基因组DNA）。
④ 酚氯仿抽提或柱纯化（可选）。
⑤ 如果使用10×彩色缓冲液进行酶切反应，得到的产物可以直接进行上样电泳。
⑥ 酶切产物电泳如有“拖带”现象，建议将产物80℃孵育20 min，或者加入6× DNA Loading Buffer with SDS，然后再进行电泳。
2. 双酶切或多酶切
① 每种快速内切酶的用量为1 μL，并根据需要适当扩大反应体系。
② 所有快速内切酶的体积总和不得超过总反应体系的1/10。
③ 如果所用的几种快速内切酶的最适反应温度不同，应先以最适温度低的酶开始酶切，再添加最适温度较高的酶，在其最适反应温度下进行酶切反应。
3. 适用于质粒的扩大反应体系
组分 | 1 μg DNA | 2 μg DNA | 3 μg DNA | 4 μg DNA | 5 μg DNA |
DNA | 1 μg | 2 μg | 3 μg | 4 μg | 5 μg |
BamHI | 1 μL | 2 μL | 3 μL | 4 μL | 5 μL |
10×缓冲液或10×彩色缓冲液 | 2 μL | 2 μL | 3 μL | 4 μL | 5 μL |
总体积 | 20 μL | 20 μL | 30 μL | 40 μL | 50 μL |
注：如果总反应体系大于20 μL，应适当增加温育时间，尽量使用水浴、金属浴或沙浴。
不同DNA中的酶切位点数量
λDNA | ΦX174 | pBR322 | pUC57 | pUC18/19 | SV40 | M13mp18/19 | Adeno2 |
5 | 0 | 1 | 1 | 1 | 1 | 1 | 3 |
甲基化修饰影响
Dam | Dcm | CpG | EcoKI | EcoBI |
无影响 | 无影响 | 无影响 | 无影响 | 无影响 |
Aladdin BamHI is a genetically engineered recombinant fast restriction endonuclease designed for rapid digestion of plasmid DNA, PCR products, and genomic DNA. The enzyme exhibits excellent activity in either the universal 10× Buffer or 10× Color Buffer, enabling complete digestion within 5–15 minutes.
The 10× Color Buffer contains red and yellow tracking dyes, allowing the digestion products to be loaded directly onto agarose gels for electrophoresis. In a 1% agarose gel, the red dye migrates at a rate comparable to that of a 2500 bp double-stranded DNA fragment, while the yellow dye migrates at a rate comparable to that of a 10 bp double-stranded DNA fragment.
Contents & Storage
Component No. | Component Name | Appearance | 500 rxns | Storage |
L358964-A | BamHI | Liquid | 500 μL | -20 ℃ |
L358964-B | 10×Buffer | Liquid | 2×1 mL | -20 ℃ |
L358964-C | 10×Color Buffer | Liquid | 2×1 mL | -20 ℃ |
Recommended Reaction Conditions
1× Buffer;
Incubate at 37 °C;
Prepare the reaction mixture according to the "Rapid DNA Digestion Protocol."
Inactivation Conditions
Heat inactivation is not applicable. Use phenol-chloroform extraction or column purification to remove the enzyme.
Quality Control
1. Functional Activity Assay
At 37 °C, 1 μL of BamHI completely digests 1 μg of λDNA within 15 min in a 20 μL reaction mixture containing the universal buffer.
2. Prolonged Incubation Assay
At 37 °C, 1 μL of BamHI is incubated with 1 μg of λDNA for 3 h in a 20 μL reaction mixture containing the universal buffer. No nonspecific substrate degradation caused by contaminating nucleases or star activity is detected. Prolonged digestion beyond this incubation period may result in star activity.
3. Digestion-Ligation-Redigestion Assay
DNA substrates are digested with a 10-fold excess of BamHI at 37 °C. After purification, more than 95% of the digested DNA fragments can be religated using T4 DNA Ligase (Fast) at 22 °C. Following recovery of the ligation products, more than 95% can be cleaved again using the same restriction endonuclease.
4. Nonspecific Endonuclease Activity Assay
At 37 °C, 1 μL of BamHI is incubated with 1 μg of supercoiled plasmid DNA for 4 h in a 20 μL reaction mixture containing the universal buffer. Subsequent agarose gel electrophoresis analysis shows that less than 10% of the plasmid DNA is converted into nicked or linear forms.
5. Blue-White Screening Assay
A specific vector containing the lacZα gene and a single restriction site within this gene is digested with 1 μL of BamHI. Following religation, the DNA products are transformed into competent Escherichia coli cells and plated onto LB agar containing X-gal, IPTG, and the appropriate antibiotic.
Successful religation restores functional β-galactosidase expression, resulting in blue colonies. In contrast, DNA fragments that fail to religate due to restriction-end degradation or other factors produce white colonies. For BamHI, the proportion of white colonies should be less than 1%.
Instructions for Use
1. Rapid DNA Digestion Protocol
① Prepare the reaction mixture on ice in the recommended order of reagent addition as follows:
Component | Plasmid DNA | PCR Product | Genomic DNA |
ddH₂O | 15 μL | 16 μL | 30 μL |
10× Buffer or 10× Color Buffer | 2 μL | 3 μLᵃ | 5 μL |
Substrate DNA | 2 μL (up to 1 μg) | 10 μL (approximately 0.2 μg) | 10 μL (5 μg) |
BamHI | 1 μL | 1 μL | 5 μL |
Total Volume | 20 μL | 30 μL | 50 μL |
Note a: This reaction system is suitable for the digestion of purified PCR products. Unpurified PCR products exhibit a certain degree of ionic strength; therefore, the volume of 10× Buffer may be reduced to 2 μL as appropriate. However, residual DNA polymerase may possess exonuclease activity that can affect the digestion products. Therefore, if subsequent cloning or related procedures are required, purification of PCR products before restriction digestion is recommended.
② Mix gently by pipetting up and down or tapping the tube (do not vortex), then briefly centrifuge to collect droplets from the tube walls.
③ Incubate at 37 °C for 15 min (plasmid DNA), 15–30 min (PCR products), or 30–60 min (genomic DNA).
④ Perform phenol-chloroform extraction or column purification (optional).
⑤ If 10× Color Buffer is used for the digestion reaction, the resulting products can be loaded directly onto an agarose gel for electrophoresis.
⑥ If smearing or trailing bands are observed during electrophoresis of the digestion products, incubate the products at 80 °C for 20 min or add 6× DNA Loading Buffer with SDS before electrophoresis.
2. Double or Multiple Restriction Digestion
① Use 1 μL of each fast restriction endonuclease and scale up the reaction volume as necessary.
② The combined volume of all fast restriction endonucleases must not exceed one-tenth of the total reaction volume.
③ If the restriction endonucleases have different optimal reaction temperatures, initiate digestion with the enzyme requiring the lower optimal temperature. Subsequently, add the enzyme requiring the higher optimal temperature and perform the digestion at its optimal reaction temperature.
3. Scaled-Up Reaction Systems for Plasmid DNA
Component | 1 μg DNA | 2 μg DNA | 3 μg DNA | 4 μg DNA | 5 μg DNA |
DNA | 1 μg | 2 μg | 3 μg | 4 μg | 5 μg |
BamHI | 1 μL | 2 μL | 3 μL | 4 μL | 5 μL |
10× Buffer or 10× Color Buffer | 2 μL | 2 μL | 3 μL | 4 μL | 5 μL |
Total Volume | 20 μL | 20 μL | 30 μL | 40 μL | 50 μL |
Note: If the total reaction volume exceeds 20 μL, the incubation time should be extended appropriately. The use of a water bath, dry block heater, or sand bath is recommended whenever possible.
Number of Restriction Sites in Different DNA Substrates
λDNA | ΦX174 | pBR322 | pUC57 | pUC18/19 | SV40 | M13mp18/19 | Adeno2 |
5 | 0 | 1 | 1 | 1 | 1 | 1 | 3 |
Effects of DNA Methylation
Dam | Dcm | CpG | EcoKI | EcoBI |
No Effect | No Effect | No Effect | No Effect | No Effect |
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