PdmI (XmnI)

Blue Ice

Molecular Biology Grade

5'-G A A N N↓N N T T C-3'
3'-C T T N N↑N N A A G-5'

Source:
Pseudomonas diminuta Auk 5–324

Concentration:
10u/ul

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

Storage Buffer:
10mM Tris-HCl, pH 7.5, 100mM KCl, 1mM DTT, 5mM MgCl2, 0.2mg/ml BSA, 50% glycerol.

Supplied with:
R1625: Restriction Enzyme Buffer A, 10X: Dilute to 1X for use.
1X buffer composition is 33mM Tris-acetate pH 7.9, 10mM magnesium acetate, 66mM potassium acetate, 0.1mg/ml BSA

Incubation Temperature:
37°C

Thermal Inactivation:
PdmI is inactivated by incubation at 65°C for 20 minutes.

Dilution Buffer:
10mM Tris-HCl, pH 7.4, 100mM KCl, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA, 50% glycerol.

Enzyme Properties:
Methylation Effects on Digestion:
Dam: Never overlaps - no effect
Dcm: Never overlaps - no effect
CpG: May overlap - cleavage impaired
EcoKI: Never overlaps - effect not determined
EcoBI: May overlap - effect not determined

Stability during Prolonged Incubation:
A minimum of 0.5 units of enzyme is required for complete digestion of 1ug of lambda DNA in 16 hours at 37°C.

Digestion of Agarose-embedded DNA:
A minimum of 10 units of enzyme is required for complete digestion of 1ug of agarose-embedded lambda DNA in 16 hours.

Number of Recognition Sites in DNA:
Lambda: 24
PhiX174: 3
pBR322: 2
pUC57: 1
pUC18/19: 1
pTZ19R/U: 1
M13mp18/19: 2

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

Ligation/Recutting Assay:
The ligation and recleavage assay was replaced with L0 test after validating experiments showed L0 test ability to trace nuclease and phosphatase activities with sensitivity that is higher than L/R by a factor of 100.

Labeled Oligonucleotide Assay:
No detectable degradation of a single-stranded or double-stranded labeled oligonucleotide was observed after incubation with 10 units of Pdml for 4 hours.

Protocol for Digestion:
Add:
Nuclease free water: 16ul
R1625: 2ul
DNA (0.5-1ug/ml): 1ul
Pdml: 0.5-2ul
Mix gently and spin down for a few seconds. Incubate at 37ºC for 1-2 hours.
The digestion reaction may be scaled either up or down.

Protocol for Digestion of PCR Products Directly after Amplification:
Add:
PCR Reaction Mixture: 10ul (~0.1-0.5ug of DNA)
Nuclease free water: 18ul
R1625: 2ul
Pdml: 1-2ul
Mix gently and spin down for a few seconds.
Incubate at 37ºC for 1-16 hours.

Storage and Stability:
May be stored at 4°C. For long-term storage, aliquot and store at -20°C. Aliquots are stable for 6 months after receipt. For maximum recovery of product, centrifuge the original vial prior to removing the cap. Further dilutions can be made in assay buffer.