NcoI

Blue Ice

Molecular Biology Grade

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

Source:
Nocardia corallina

Concentration:
10u/ul

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

Supplied With:
R1625: Restriction Enzyme Buffer A, 10X Supplied as a liquid. 1x contains 33mM Tris-acetate, 10mM magnesium acetate, 66mM potassium acetate, 0.1mg/ml BSA.

Storage Buffer:
Supplied as a liquid in 10mM Tris-HCl, pH 7.5, 50mM KCl, 1mM DTT, 0.1mM EDTA, 0.2mg/ml BSA, 50% glycerol.

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

Double Digests:
Restriction Enzyme Buffer A, 10X, is provided to simplify buffer selection for double digests. Diluted to 1X, the formulation is 33mM Tris-acetate, pH 7.9, 10mM magnesium acetate, 66mM potassium acetate, 0.1mg/ml BSA. Incubate at 37°C.

Overdigestion Assay:
No detectable change in the specific fragmentation pattern is observed after 80-fold overdigestion (5u/ug lambda DNA x 16 hours) with NcoI.

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

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

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

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

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

Methylation Effects on Digestion:
Dam, Dcm, CpG, EcoKI, EcoBI: Never overlaps, no effect

Compatible Ends:
Afl III, PscI, BtgI, Eco130I, FatI, Pag I

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

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

Protocol for Digestion:
Add Nuclease free water (16ul)
R1625 (2ul)
DNA (0.5-1ug/ml) (1ul)
N0554 (0.5-2ul)
Mix gently and spin down for a few seconds. Incubate at 37ºC for 1-16 hours

Protocol for Digestion Directly after Amplification:
Add PCR Reaction Mixture (10ul) (~0.1-0.5ug of DNA))
Nuclease free water (18ul)
R1625 (2ul)
N0554 (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 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.