BsaI

Cat. No.: L359050

グレード＆純度 Recombinant, for DNA synthesis, Suitable for molecular biology, EnzymoPure™, For In Vitro Transcription, for DNA and RNA applications, 20 U/μl; expressed in E.coli

Expression system

E. coli

Bioactivity

20 U/μl

概要

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

仕様

Product Name

BsaI

同義語

Eco31I | BsaI | Bsa I | Eco31I | Bso31I | BspTNI

等級

for DNA and RNA applications, for DNA synthesis, For In Vitro Transcription, Recombinant, Suitable for molecular biology, EnzymoPure™

仕様と純度

Recombinant, for DNA synthesis, Suitable for molecular biology, EnzymoPure™, For In Vitro Transcription, for DNA and RNA applications, 20 U/μl; expressed in E.coli

生物活性

20 U/μl

分子タイプ

酵素

Product Line

生物製品

保管と配送

集中

20 U/μl; expressed in E.coli

保管条件

Store at -20°C,Avoid repeated freezing and thawing

入荷

Ice chest + Ice pads

安定性と保管

Store at 2-8℃ short term (1-2 weeks). Store at -20℃ long term (24 months). Upon receipt, it is recommended to aliquot.

ユニットの定義

One unit (U) is defined as the amount of enzyme required to completely digest 1 μg of pPIC9K (Dcm⁻) DNA in a 50 μL reaction system at 37 °C within 1 hour.