Restriction endonucleases

Description

Product Details

ApaL I is a restriction enzyme purified from Acetobacter pasteurianus (ATCC 12875).

Reagents supplied: 10x R1 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 ApaL 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	100	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: No

Methylation Sensitivity:

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

Unit calculation assay conditions:

• 1x R1 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
• 200 μg/ml BSA
• 50% glycerol
• Store at -20^oC

Non-specific nuclease activity: 100 units of ApaL 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 ApaL I, greater than 98% of the DNA fragments can be ligated and recut with this enzyme.