PaeI (SphI)

Blue Ice

Molecular Biology Grade

Sequence:
5'-G C A T G^C-3'
3'-C^G T A C G-5'

Source:
Pseudomonas aeruginosa

Concentration:
10u/ul

Unit Definition:
One unit is defined as the amount of PaeI required to digest 1ug of lambda DNA fragments in 1 hr at 37°C in 50ul assay buffer.

Diluent Buffer:
Supplied as a liquid in 10mM Tris-HCl, pH 7.4, 100mM KCl, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA, 50% glycerol.

Storage Buffer:
10mM potassium phosphate (pH 7.0 at 25°C), 50mM KCl, 1mM DTT, 0.1mM EDTA, 0.5mg/ml BSA, 0.15% Triton X-100 and 50% glycerol.

Supplied with:
R1625: Supplied as a liquid in 33mM Tris-acetate, 10mM magnesium acetate, 66mM potassium acetate, 0.1mg/ml BSA, pH 7.9 at 37ºC.
R1625-01: Restriction Enzyme Buffer B, 10X: Supplied as a liquid 10mM Tris-HCl, pH 7.5 at 37°C, 10mM magnesium chloride, 0.1mg/ml BSA. Incubate at 37°C

Double Digests:
R1625 is provided to simplify buffer selection for double digests. 98% of PaeI, works well in a 1x or 2x.

Overdigestion Assay:
No detectable change in the specific fragmentation pattern is observed after 160-fold overdigestion (10u/ug lambda DNA x 16 hours) with PaeI.

Ligation Recutting Assay:
After 50-fold overdigestion (3u/ug DNA x 17 hours) with PaeI, more than 95% of the DNA fragments can be ligated at a 5'-termini concentration of 0.06uM. More than 95% of these can be recut.

Labeled Oligonucleotide (LO) Assay:
No detectable degradation of a single-stranded and double-stranded labeled oligonucleotide was observed after incubation with 10 units of PaeI for 4 hours.

Blue White Cloning Assay:
pUC57 was incubated with 10 units of PaeI for 16 hours. After religation and transformation <1% of white colonies were detected.

Methylation Effects:
Dam:, Dcm, EcoKI: never overlaps-no effect
CpG: May overlap- no effect
EcoBI: may overlap- blocked

Stability during Prolonged Incubation:
A minimum of 0.1 units of PaeI is required for complete digestion of 1ug of lambda DNA in 16 hours at 37°C.

Thermal Inactivation:
PaeI is inactivated by incubation at 65°C for 20min.

Digestion of Agarose-embedded DNA:
A minimum of 5 units of PaeI is required for digestion of 1ug of agarose-embedded lambda DNA in 16 hours.

Number of Recognition Sites in DNA:
Lambda6
pBR3221
phiX174 0
pUC57, pUC18/19, pTZ19R/U, M13mp18/19