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 37oC in a total reaction volume of 50 μl.
Standard reaction conditions: In order to overcome DNA source variability, a ~10 fold overdigest is suggested:
10U Nae I
10x EQ buffer
Sterile ultrapure water
Up to 50 μl
Speed Digest reaction: Incubate for 15 minutes at 37oC as in standard reaction in 1x R1 buffer ( 10 mM Tris-HCl (pH 7.9 @ 25oC), 10 mM MgCl2, 100 μg/ml BSA) or 1x eQ buffer (20 mM Tris-acetate (pH 7.9 @ 25oC), 10 mM Mg-acetate, 50 mM K-acetate, 100 μg/ml BSA).
Activity(%) in EnzyQuest buffers-Double digest tips
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: 65oC for 20 minutes
dam methylation: Not sensitive
dcm methylation: Not sensitive
CpG methylation: Blocked
Unit calculation assay conditions:
1x R1 buffer
Incubation at 25oC
50 mM KCl,
10 mM Tris-HCl (pH 7.4)
0.1 mM EDTA
1 mM dithiothreitol
200 μg/ml BSA
Store at -20oC
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 37oC.
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.
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