Tanım
BspEI is a Type IIP restriction enzyme originally isolated from Bacillus spp. and produced via recombinant expression and purification in Escherichia coli. BspEI recognizes and cleaves the sequence TCCGGA, generating a 5′-overhanging cohesive end with a 4-nucleotide protrusion. BspEI requires its dedicated buffer, Cut Buffer C, and is not compatible with the universal buffer FuniCutTM Buffer.
Specifications
|
Cat.No. |
15230ES76 |
|
Unit Size |
500 U |
|
Recognition Site |
5'-T↓CCGGA-3' 3'-AGGCC↑T-5' |
|
Reaction Conditions |
1×Cut Buffer C; incubate at 37°C |
|
Inactivation |
Incubate at 80°C for 20 min |
|
Unit Definition |
One unit (U) is defined as the amount of enzyme required to completely digest 1 μg of λ DNA in 50 μL reaction volume in 1 hour at 37°C. |
|
Isoschizomer |
Kpn2I, MroI, AccIII, Aor13HI, Bsp13I, BseAI |
Components
|
Components No. |
Name |
15230ES76 |
|
15230-A |
BspEI (10 U/μL) |
50 μL |
|
15230-B |
10× Cut Buffer C |
1 mL |
Storage
This product should be stored at -25~-15℃ for 2 years.
Instructions
1. DNA Digestion Protocol
1)Prepare the reaction mixture on ice following the recommended component addition order below:
|
Components |
Volume |
|
ddH2O |
To 50 μL |
|
10× Cut Buffer C |
5 μL |
|
Substrate DNA* |
1 μg |
|
BspEI (10 U/μL) |
1 μL |
|
Total |
50 μL |
[Note]: The DNA substrate must be free of phenol, chloroform, ethanol, EDTA, detergents, or high salt concentrations, as these may inhibit BspEI activity.
2)Gently mix by pipetting up and down or flicking the tube wall (do not vortex), then briefly centrifuge to collect droplets from the tube walls.
3)Incubate at 37°C for 15 min ~ 3 h.
4)Inactivate the enzyme by incubating at 80°C for 20 min to stop the reaction, or terminate by purification using a spin column or phenol/chloroform extraction.
2. Number of Recognition Sites in Different Genomes
|
λDNA |
ΦX174 |
pBR322 |
pUC57 |
pUC18/19 |
SV40 |
M13mp18/19 |
Adeno2 |
|
24 |
0 |
1 |
0 |
0 |
0 |
0 |
8 |
3. Effect of Methylation
|
Dam |
Dcm |
CpG |
EcoKI |
EcoBI |
|
Cleavage blocked |
No effect |
Cleavage affected |
No impact |
No impact |
4. Activity in Different Reaction Buffers*
|
Reaction buffer |
FuniCutTM Buffer(Yeasen) |
Thermo Scientific FastDigest Buffer |
NEB CutSmart® Buffer |
Takara QuickCut™ Buffer |
|
Activity |
<10% |
50% |
<10% |
50% |
Notes
1. This product is for research use only.
2. Please operate with lab coats and disposable gloves，for your safety.
3. The volume of enzyme added should not exceed 10% of the total reaction volume to avoid star activity caused by excess glycerol in the enzyme storage buffer.
4. Additives in the restriction enzyme storage buffer (e.g., glycerol, salts) can interact with contaminants in the DNA sample (e.g., salts, EDTA, ethanol). The smaller the reaction volume, the stronger the inhibitory effect on digestion.
Documents
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Sorgu
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