|
Source
: Frankia species Eul1b (NRRL 18528)
Compatible ends
(CCGG…3') & Isoschizomers
: Rig I
Methylation Sensitivity
: Not sensitive to dam and dcm methylation
: Blocked by CpG methylation
Unit definition :
One Unit is defined as the amount of Fse I required to completely digest 1 ㎍ of Adenovirus-2 DNA in one hour at 37℃ in 50 ㎕ assay mixture.
Typical Reaction Conditions
: Add 5 ㎕ of 10X Reaction Buffer 'E'[final conc.: 20 mM Tris-acetate (pH 7.9 @ 25℃), 50 mM Potassium acetate, 10 mM Magnesium acetate, 1 mM DTT], 1~2 ㎕ of DNA(0.5~1.0 ㎍/㎕), 5 ㎕ of 10X BSA(final conc.: 100 ㎍/㎖), 1 ㎕ of Fse I and 37~38 ㎕ of sterile water in 50 ㎕ reaction mixture. Incubate at 37℃.
Heat Inactivation
: 65℃ for 20 min.
Activity in BeamsBio™ Buffer System
|
Rxn Buffer |
A |
B |
C |
D |
E |
|
Activity(%) |
75~100 |
75~100 |
<10 |
<10 |
75~100 |
|
Activity in a complete PCR Mix : less than 10%
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 Fse I. And then incubate at 37℃ for one hour. Relative activity is determined by gel electrophoresis.
Storage Conditions
: 10 mM Tris-HCl(pH 7.4), 200 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 200 ㎍/㎖ BSA, and 50% glycerol. Store at -70℃.
No. of Cleavage Sites
|
λ
|
Ad2
|
pUC18
|
φX174
|
pBR322
|
M13mp18
|
SV40
|
|
0
|
3
|
0
|
0
|
0
|
0
|
0
|
Quality control
Overdigestion: The same band pattern as a digestion in one unit of Fse I for one hour was showing after incubation of 1 ㎍ of Ad-2 DNA with units of Fse I for 16 hr in 50 ㎕ of reaction mixture at 37℃ as determined by agarose gel electrophoresis.
Ligation-Recut: After a 10-fold digestion for one hour, % of Ad-2 DNA fragments were ligated with T4 DNA ligase at 16℃ and % of the ligated fragments could be recut with Fse I.
|
|
Notes
- Fse I is one of the least stable restriction enzymes when stored at -20℃.
We recommend storage at -70℃ if it is to be stored for longer than 30 days.
- Fse I is strongly inhibited by certain salts, including NaCl or ammonium acetate. If you perform alcohol precipitation
before digestions with Fse I, use sodium acetate (or potassium acetate) as the salt in the precipitation.
|
|
References
Nelson, J.M., Miceli, S.M., Lechevalier, M.P., Roberts, R.J. Nucleic Acid Res. 18: 2061-2064 (1990)
|
