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FAQ

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What is star activity?

A: Some restriction enzymes cleave similar sequence to the recognition sequence or nonspecifically in unsuitable reaction conditions which referred as star activity.   


Conditions that Contribute to Star Activity 

  1) High glycerol concentration [> 5% v/v] 

  2) High units to μg of DNA ratio [Varies with each enzyme, usually >100 units/μg] 

  3) Low ionic strength [< 25 mM] 

  4) High pH [> pH 8.0] 

  5) Presence of organic solvents 

      [DMSO, ethanol, ethylene glycol, dimethylacetamide, dimethylformamide, sulphalane] 

  6) Substitution of Mg2+ with other divalent cations [Mn2+, Cu2+, Co2+, Zn2+]


Inhibiting Star Activity 

 If you are concerned about star activity, we recommend the following guidelines. 

  1) Use as few units as possible to get a complete digestion. This avoids overdigestion 

      and reduces the final glycerol concentration in the reaction. 

  2) Make sure the reaction is free of any organic solvents such as alcohols which might 

      be present in the DNA preparation. 

  3) Raise the ionic strength of the reaction buffer to 100-150 mM (provided the enzyme

       is not inhibited by high salt). 

  4) Lower the pH of the reaction buffer to pH 7.0. 

  5) Use Mg2+ as the divalent cation


Enzynomics has developed EZ-CleanCut™ restriction enzymes with reduced star activity for more accurate experiments.

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