Using these guidelines to prevent star activity:
Maintain the pH between 7.2 and 8. The exact pH will depend on the restriction enzyme you’re using. High pH above 8 is one of the causes of star activity.
Use the minimum amount of restriction enzyme needed for complete digestion. This will prevent over-digestion of the DNA and maintain a lower concentration of glycerol in the reaction. High glycerol concentration of >5% causes star activity. The recommended amount is 1 unit restriction enzyme for every microgram of DNA, assuming you’ll incubate the reaction for one hour. If you’re using supercoiled DNA, you’ll need to add 3 to 5 times more enzyme than you would when using linear DNA.
Presence of organic solvents that may have carried over during DNA purification can also cause star activity. To prevent this, make sure the reaction is free from any type of organic solvent.
The ionic strength of the reaction buffer should be increased to between 50mM and 150mM. This is provided high salt is not inhibiting the enzyme.
Use Mg2+ as the preferred divalent cation in the reaction buffer.
Use the minimum reaction time necessary for complete digestion to prevent star activity from prolonged incubation and evaporation.