Restriction endonucleases

Description

Product Details

NaeI is a restriction enzyme purified from Streptomyces species.

Reagents supplied: 10x R1 and 10x EQ buffer

Unit definition: One unit is the amount of enzyme required to digest 1 μg of pBR322 DNA in 60 min at 37^oC in a total reaction volume of 50 μl.

Digest Guidelines

• Standard reaction conditions: In order to overcome DNA source variability, a ~10 fold overdigest is suggested:

10U Nae I	1μl
10x EQ buffer	5μl
DNA substrate	1μg
Sterile ultrapure water	Up to 50 μl

• Speed Digest reaction: Incubate for 15 minutes at 37^oC as in standard reaction in 1x R1 buffer ( 10 mM Tris-HCl (pH 7.9 @ 25^oC), 10 mM MgCl2, 100 μg/ml BSA) or 1x eQ buffer (20 mM Tris-acetate (pH 7.9 @ 25^oC), 10 mM Mg-acetate, 50 mM K-acetate, 100 μg/ml BSA).

• Activity(%) in EnzyQuest buffers -Double digest tips

R1	R2	R3	R4	EQ
100	25-50	25	<10	100

Reactions were performed as for unit definition reaction. The above data could be used as guideline for double digest reactions. Our enzymes are also active and compatible in buffer systems from other vendors.

Heat inactivation: 65^oC for 20 minutes

Methylation Sensitivity:

• dam methylation: Not sensitive
• dcm methylation: Not sensitive
• CpG methylation: Blocked

Unit calculation assay conditions:

• 1x R1 buffer
• pBR322 DNA
• Incubation at 25^oC

Storage conditions:

• 50 mM KCl,
• 10 mM Tris-HCl (pH 7.4)
• 0.1 mM EDTA
• 1 mM dithiothreitol
• 200 μg/ml BSA
• 50% glycerol
• Store at -20^oC

Non-specific nuclease activity: 50 units of Nae I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of pBR322 at 37^oC.

Re-ligation and recut: After 10-fold overdigestion with Nae I, greater than 80% of the DNA fragments can be ligated and recut with this enzyme.

Note: Nae I exhibits site preferences. pBR322 contains four Nae I recognition sequences. Two of these sites are readily cleaved, one is cleaved moderately slowly, and the fourth is cleaved 50-fold more slowly.