Restriction endonucleases

Description

Product Details

StyI is a restriction enzyme purified from E.coli WA921/pST27 hsd+.

Reagents supplied: 10x R3 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 Sty I	1μl
10x eQ buffer	5μl
DNA substrate	1μg
Sterile ultrapure water	Up to 50 μl
Incubate for 60 min at 37ºC

• Speed Digest reaction: Incubate for 15 minutes at 37^oC as in standard reaction in 1x R3 buffer (100 mM NaCl, 50 mM Tris-HCl (pH 7.9 @ 25°C), 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
25-50	75-100	100	75-100	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: 65ºC for 20 minutes 

Methylation Sensitivity: 

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

Unit calculation assay conditions:  

• 1x R3 buffer 
• Lambda DNA  
• Incubation at 65ºC  

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 -20ºC 

Non-specific nuclease activity: 50 units of Sty I do not produce any unspecific clevage products after 16 hrs incubation with 1 μg of λ DNA at 37°C. 

Re-ligation and recut: After 50-fold overdigestion with Sty I, greater than 98% of the DNA fragments can be ligated and recut with this enzyme. 

Star activity: Conditions of low ionic strength, high enzyme concentration, glycerol concentration >5%, or pH >8.0 may result in star activity