Restriction endonucleases

Description

Product Details

ScaI is a restriction enzyme purified from Streptomyces caespitosus.

Reagents supplied: 10x ScaI buffer and 10x eQ buffer

Unit definition: One unit is the amount of enzyme required to digest 1 μg of Lambda 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 Sca I	1μl
10x ScaI 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 ScaI buffer (100 mM KCl, 10 mM Bis-Tris-Propane (pH 7.0 @ 25°C), 10 mM MgCl2, 100 μg/ml BSA) or 5 units in 2x eQ buffer (40 mM Tris-acetate (pH 7.9 @ 25^oC), 20 mM Mg-acetate, 100 mM K-acetate, 200 μg/ml BSA) .

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

R1	R2	R3	R4	EQ
<10	25-50	100	75-100	75-100

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: 80^oC for 20 minutes

Methylation Sensitivity:

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

Unit calculation assay conditions:

• 1x ScaI buffer
• Lambda DNA
• Incubation at 37^oC

Storage conditions:

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

Non-specific nuclease activity: 600 units of Sca I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of Lambda DNA at 37^oC.

Re-ligation and recut: After 100-fold overdigestion with Sca I, greater than 90% of the DNA fragments can be ligated and recut with this enzyme.

Star activity: Large excess of the enzyme may results in the appearance of star activity.