Restriction endonucleases

Description

Product Details

PspP I is a restriction enzyme purified from Psychrobacter immobilis TA137.

Reagents supplied: 10x R2 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 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 PspP 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 R2 buffer (50 mM NaCl, 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
50-75	100	50	25-50	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: 55^oC for 15 minutes

Methylation Sensitivity:

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

Unit calculation assay conditions:

• 1x R2 buffer
• Lambda DNA
• Incubation at 25^oC

Storage conditions:

• 50 mM NaCl,
• 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: 80 units of PspP 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 PspP I, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.

Note: Incubation at 37^oC results in 60% activity.