|
Source : Bacillus stearothermophilus X1
Compatible ends (NNNN…3')
BstX I has no compatible ends to other known restriction enzymes.
Isoschizomers
: BstHZ55 I
Methylation Sensitivity
: Not sensitive to dam and CpG methylation
: Blocked by overlapping dcm methylation
Unit definition : One Unit is defined as the amount of BstX I required to completely digest 1 ㎍ of λ DNA in one hour at 50℃ in 50 ㎕ assay mixture.
Typical Reaction Conditions : Add 5 ㎕ of 10X Reaction Buffer 'D'[final conc.: 6 mM Tris-HCl(pH 7.9 @ 37℃), 6 mM MgCl2, 150 mM NaCl, 1 mM DTT], 1~2 ㎕ of DNA(0.5~1.0 ㎍/㎕), 1 ㎕ of BstX I and 42~43 ㎕ of sterile water in 50 ㎕ reaction mixture. Incubate at 50℃.
Heat Inactivation : 65℃ for 20 min.
Activity in BeamsBio™ Buffer System
|
Rxn Buffer |
A |
B |
C |
D |
|
Activity(%) |
<10 |
10~50 |
10~50 |
75~100 |
|
Activity in a complete PCR Mix : 10~50%
Conditions: [10 mM Tris-HCl(pH 8.3 @ 25℃), 50 mM KCl, 1.5 mM MgCl2, 2 pmol of primers, 200 μM dNTP Mix, 2.5 U Taq DNA polymerase and 1 ㎍ of DNA(template or substrate) in 50 ㎕ of reaction volume] After 30 amplication cycles, add 5 units of BstX I. And then incubate at 50℃ for one hour. Relative activity is determined by gel electrophoresis.
Storage Conditions : 10 mM Tris-HCl (pH 7.5 @ 25℃), 400 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.15% Triton™ X-100, 100 ㎍/㎖ BSA, and 50% glycerol. Store at -20℃.
No. of Cleavage Sites
|
λ |
Ad2 |
pUC18 |
φX174 |
pBR322 |
M13mp18 |
SV40 |
|
13 |
10 |
0 |
3 |
0 |
0 |
1 |
Quality control
Overdigestion: The same band pattern as a digestion in one unit of BstX I for one hour was showing after incubation of 1 ㎍ of λ DNA with units of BstX I for 16 hr in 50 ㎕ of reaction mixture at 50℃ as determined by agarose gel electrophoresis.
Ligation-Recut: After a 10-fold digestion for one hour, % of λ DNA fragments were ligated with T4 DNA ligase at 16℃ and % of the ligated fragments could be recut with BstX I. |
