PdiI (NaeI)

Blue Ice

Molecular Biology Grade

5'-G C C^G G C-3'
3'-C G G^C C G-5'

Concentration:
10u/ul

Source:
Pseudomonas diminuta Mck 33–321

Stability during Prolonged Incubation:
A minimum of 0.5 units of P3121 is required for complete digestion of 1ug of DNA in 16 hours at 37°C.

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

Digestion of Agarose-embedded DNA:
A minimum of 20 units of P3121 is required for complete digestion of 1ug of agarose- embedded pBR322 DNA in 16 hours.

Unit Definition:
One unit is defined as the amount of P3121 required to digest 1ug of pBR322 DNA-NdeI fragments in 1 hour at 37°C in 50ul of reaction bu

Number of Recognition Sites in DNA:
Lambda: 1
PhiX174: 0
pBR322: 4
pUC18/19: 0
pUC57: 0
pTZ19R/U: 1
M13mp18/19: 1

Recommended Protocol:
Add: Nuclease-free water 16ul
R1625: 2ul
DNA: 1ul
P3121: 0.5-2ul
1. Mix gently and spin down for a few seconds.
2. Incubate at 37ºC for 1-16 hours.

Protocol for Digestion of PCR after Amplification:
Add: PCR mixture: 10ul
Nuclease-free water: 18ul
R1625: 2ul
P3121: 1-2ul
1. Mix gently and spin down for a few seconds.
2. Incubate at 37ºC for 1-16 hours.

Menthylation Effects on Digestion:
No effect on Dam, Dcm, EcoKl and EcoBl.
Overlaps and blocks CpG.

Supplied with:
R1625: Restriction Enzyme Buffer A, 10X:
Dilute to 1X for use. 1X buffer composition is 33mM Tris-acetate pH 7.9 at 37°C, 10mM magnesium acetate, 66mM potassium acetate, 0.1mg/ml BSA.

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

Storage Buffer:
Supplied as a liquid in 10mM Tris-HCl, pH 7.4, 500mM KCl, 1mM DTT, 0.1mM EDTA, 0.2mg/ml BSA, 0.15% Triton X-100, 50% glycerol.

Overdigestion Assay:
No detectable change in the specific fragmentation pattern is observed after 160-fold overdigestion (10u/ug lambda DNA x 16 hours) with P3121.

Ligation and Recleavage (L/R) Assay:
The ligation and recleavage assay was replaced with L0 test after validating experiments showed L0 test abililty to trace nuclease and phophatase activities with senstivity that is higher than L/R by a fctor of 100.

Labeled Oligonucleotide (LO) Assay:
No detectable degradation of a single-stranded and double-stranded labeled oligonucleotide occurred during incubation with 10 units of Pdil for 4 hours.

Blue/White Cloning Assay:
The B/W assay was replaced with L0 test after validating experiments showed L0 test ability to detect nuclease and phophatase activities with sensitivity that equals to that of B/W test.

Methylation Effects:
No effect on Dam, Dcm, EcoKl and EcoBl. Overlaps and blocks CpG.

Storage and Stability:
May be stored at 4°C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20°C. Aliquots are stable for 6 months after receipt at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.