Restriction endonucleases

Description

Product Details

PstI is a restriction enzyme purified from a recombinant E.coli strain.

Reagents supplied: 10x PstI 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 Pst 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 PstI buffer (100 mM NaCl, 50 mM Tris-HCl (pH 7.4 @ 25oC), 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
10-25	50-75	75-100	50-75	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 PstI buffer
• Lambda DNA
• Incubation at 37^oC

Storage conditions:

• 200 mM NaCl,
• 10 mM Tris-HCl (pH 7.4)
• 0.1 mM EDTA
• 1 mM dithiothreitol
• 0.15% Triton X-100
• 200 μg/ml BSA
• 50% glycerol
• Store at -20^oC

Non-specific nuclease activity: 200 units of Pst 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 Pst I, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.

Star activity: Conditions of high enzyme concentration or glycerol concentration >12% may result in star activity.