Restriction endonucleases

Description

Product Details

SmaI is a restriction enzyme purified from Serratia marcescens (ATCC 49779).

Reagents supplied: 10x R5 and 10x EQ buffer

Unit definition: One unit is the amount of enzyme required to digest 1 μg of Lambda DNA (Hind III digest) in 60 min at 25^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 Sma I	1μl
10x EQ buffer	5μl
DNA substrate	1μg
Sterile ultrapure water	Up to 50 μl

• Speed Digest reaction: Incubate for 15 minutes at 25^oC as in standard reaction in 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
<10	<10	<10	<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 EQ buffer
• Lambda 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: 150 units of Sma I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of Lambda DNA at 25^oC.

Re-ligation and recut: After 50-fold overdigestion with Sma I, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.