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
| Component
| Appearance
| 1KU | 5×1KU | Storage
| | L359050A | BsaI (20U/μl)
| Liquid
| 50μl
| 5×50μl
| -20℃ | | L359050B | Buffer
| Liquid
| 1ml | 5×1ml
| -20℃
| | L359050C | 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 |
|
