I am using some restriction enzymes, and according to the protocol, the amount of enzyme should not exceed 10% of the total reaction volume to avoid star activity. This activity is due to the high glycerol concentration that I would have if I used more than 10% of the enzyme (that is stored in 50% glycerol). Could you please explain to me how can glycerol induce this star activity in restriction enzymes?
My guess is that the association between high glycerol concentration and star activity is not so much about the glycerol concentration as about the enzyme concentration. Since the enzymes are prepared in a 50% glycerol solution, a high glycerol concentration goes along with an excessive enzyme concentration. Instead of adding more enzyme, resulting in star activity, you should either let the reaction go longer, or use a high-concentration restriction enzyme, or clean up your DNA so that the digestion is more efficient, or otherwise follow the guidelines in the link below:
https://www.neb.com/tools-and-resources/usage-guidelines/star-activity
Hi there,
As for any other enzyme, specificity decreases when concentration of enzyme increases (or rather ratio [enzyme]/[substrate]). Then the activity of the enzyme becomes less specific...
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