限制性内切酶简称限制酶，是一类可识别特定的脱氧核苷酸序列，并对每条链中特定部位的两个脱氧核糖核苷酸之间的磷酸二酯键进行切割的核酸内切酶。BsaI 是一 种 IIS 型限制酶，能识别非回文序列，并在识别序列外部切割产生粘性末端。
同裂酶：Eco31I, Bso31I, BspTNI
识别位点：GGTCTC(1/5)
5'...G G T C T C (N)₁ ↓...3'
3'...C C A G A G (N)₅ ↑...5'
组分表
L359050
| 组件 | 物理外观 | 1KU | 5×1KU | 储存 | | L359050A | BsaI (20U/μl)
| 液体 | 50μl
| 5×50μl
| -20℃ | | L359050B | 10×Buffer | 液体 | 1ml | 5×1ml
| -20℃
| | L359050C | 10×Color Buffer | 液体 | 1ml | 5×1ml
| -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
80℃温育 20 min。
质量控制
功能活性检测
37℃下，在 20 μl 通用反应体系中，1 μl BsaI 能够在 15 min 内完全消化 1 μg pPIC9K DNA。
超长时间温育检测
37℃下，在 20 μl 通用反应体系中，将 1 μl BsaI 与 1 μg pPIC9K DNA 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。
酶切 - 连接 - 再酶切检测
37℃下，使用 10 倍酶量的 BsaI 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。
非特异性内切酶活性检测
37℃下，在 20 μl 通用反应体系中将 1 μl BsaI 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。
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：
| 质粒 DNA | PCR 产物 | 基因组 DNA |
|---|
| ddH₂O | 15 μl | 16 μl | 30 μl | | 10× Buffer 或 10× Color Buffer | 2 μl | 3 μlᵃ | 5 μl | | 底物 DNA | 2 μl (up to 1 μg) | 10 μl (~0.2 μg) | 10 μl (5 μg) | BsaI (20U/μl)
| 1 μl | 1 μl | 5 μl | | Total | 20 μl | 30 μl | 50 μl |
|
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× Buffer 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 80℃温育 20 min 即可使酶失活，停止反应（可选）；
⑤ 如果使用 Color Buffer 进行酶切反应，得到的产物可以直接进行上样电泳。
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
| 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 | BsaI (20U/μl)
| 1 μl | 2 μl | 3 μl | 4 μl | 5 μl | | 10× Buffer 或 10× Color Buffer | 2 μl | 2 μl | 3 μl | 4 μl | 5 μl | | Total | 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 | Adexnc2 |
|---|
| 2 | 0 | 1 | 1 | 1 | 0 | 0 | 18 |
|
甲基化修饰影响
| Dam | Dcm | CpG | EcoKI | EcoBI |
|---|
| 无影响 | 剪切受影响 | 剪切受影响 | 无影响 | 无影响 |
|
Restriction endonucleases, abbreviated as restriction enzymes, are a class of endodeoxyribonucleases that recognize specific deoxyoligonucleotide sequences and cleave the phosphodiester bond between two deoxyribonucleotides at a specific position on each DNA strand. BsaI is a Type IIS restriction enzyme; it recognizes non-palindromic sequences and cuts outside the recognition sequence to generate sticky overhangs.
Isoschizomers: Eco31I, Bso31I, BspTNI
Recognition Site: GGTCTC(1/5)
5'...G G T C T C (N)₁ ↓...3'
3'...C C A G A G (N)₅ ↑...5'
Components List
L359050
| Components
| Appearance
| 1KU | 5×1KU | Storage
| | L359050A | BsaI (20U/μl)
| Liquid
| 50μl
| 5×50μl
| -20℃ | | L359050B | 10×Buffer
| Liquid
| 1ml | 5×1ml
| -20℃
| | L359050C | 10×Color Buffer
| Liquid
| 1ml | 5×1ml
| -20℃ |
|
Recommended Reaction Conditions
1× Buffer;
Incubate at 37 °C;
Refer to "Protocol for Fast DNA Digestion" for reaction setup.
Heat Inactivation
Incubation at 80℃ for 20 minutes.
Quality Control
Functional Test
A 20 μl reaction in Buffer containing 1 μg of pPIC9K DNA and 1 μl of BsaI incubated for 15 minutes at 37℃ results in complete digestion as determined by agarose gel electrophoresis.
Prolonged Incubation / Star Activity Assay
A 20 μl reaction in Buffer containing 1 μg of pPIC9K DNA and 1 μl of BsaI incubated for 3 hours at 37 ℃ results in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis. Longer incubation may result in star activity.
Ligation and Recuting
After 10-fold over-digestion with BsaI at 37 ℃ , >95% of the DNA fragments can be ligated with T4 DNA Ligase at 22℃ . Of these ligated fragments, >95% can be recut with BsaI as determined by agarose gel electrophoresis.
Non-specific Endonuclease
Activity A 20 μl reaction in Buffer containing 1 μg of supercoiled plasmid and 1 μl of BsaI incubated for 4 hours at 37℃ results in <10% conversion to the nicked or linearized form as determined by agarose gel electrophoresis.
Method of application
1. Protocol for Fast DNA Digestion
① Combine the following reaction components on ice in the order indicated:
| 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 | DNA
| 2 μl (up to 1 μg) | 10 μl (~0.2 μg) | 10 μl (5 μg) | BsaI (20U/μl)
| 1 μl | 1 μl | 5 μl | | Total | 20 μl | 30 μl | 50 μl |
|
a. For purified PCR products. If the PCR products are not purified, amount of 10× Buffer should be reduced to 2 μl due to the remaining metal ions in the unpurified PCR products. We recommend to purify PCR products before digestion if it will be used for cloning, because the exonuclease activity of some DNA polymerases may alter the end of cleaved DNA.
② Mix gently and spin down;
③ Incubate at 37°C for 15 minutes (plasmid DNA) or for 15~30 minutes (PCR product) or for 30~60 minutes (genomic DNA);
④ Optional: Inactivate the enzyme by heating for 20 minutes at 80°C;
⑤ If the Color Buffer was used in the reaction, load an aliquot of the reaction mixture directly on a gel.
2. Double and Multiple Digestion of DNA
① Use 1 μl of each enzyme and scale up the reaction conditions appropriately;
② The combined volume of the enzymes in the reaction mixture should not exceed 1/10 of the total reaction volume;
③ If the enzymes require different reaction temperatures, start with the enzyme that requires a lower temperature, then add the second enzyme and incubate at the higher temperature.
3. Scaling up Plasmid DNA Digestion Reaction
| 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 | BsaI (20U/μl)
| 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 | 20 μl | 20 μl | 30 μl | 40 μl | 50 μl |
Note: Increase the incubation time if the total reaction volume exceeds 20 μl. |
Number of Recognition Sites in DNA
| λDNA | ΦX174 | pBR322 | pUC57 | pUC18/19 | SV40 | M13mp18/19 | Adexnc2 |
|---|
| 2 | 0 | 1 | 1 | 1 | 0 | 0 | 18 |
|
Methylation Effects on Digestion
| Dam | Dcm | CpG | EcoKI | EcoBI |
|---|
No effect
| Impaired
| Impaired
| No effect
| No effect |
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