AAT Bioquest

What factors should I consider before I use restriction enzymes to cut DNA into small fragments?

Posted May 2, 2023


These are the 7 factors you should consider before using restriction enzymes to cut DNA into small fragments: 

  1. Temperature should be maintained at 37°C, which is the optimum temperature for most restriction enzymes. A few enzymes that are derived from microorganisms that thrive in extreme conditions show optimum activity at higher temperatures. 
  2. pH should be maintained between 7.2 and 8.5, which is the optimum pH range for most enzymes. Altered specificity of the restriction enzyme or star activity, may increase outside of the optimal pH range. 
  3. Choose a methylation-incompetent E.coli strain when propagating your plasmid. This will prevent any problems resulting from the inability of some enzymes to bind with methylated recognition sites. 
  4. Ensure your enzymes have at least six base pairs on either side of the recognition site to improve efficiency. This is particularly important when doing a double digest or when digesting the ends of a PCR product. 
  5. Using BSA during digestion reactions helps to stabilize restriction enzymes by protecting the enzyme from non-specific adsorption and various other detrimental factors such as intense heat, surface tension and the adverse effects of proteases
  6. Be careful to avoid extended incubation periods and sub-optimal buffer conditions such as incorrect pH, high glycerol concentration or low magnesium concentration. These mistakes can increase the likelihood of producing unexpected results such as cleaving the DNA at the wrong sites or facilitating single base substitutions. 
  7. 7. Using isoschizomers and neoschizomers can help when you need to use restriction enzymes that recognize the same target sequence but exhibit completely different properties. Isoschizomers refer to different restriction enzymes that recognize and cleave the same DNA sequence. Neoschizomers refer to restriction enzymes that recognize the same DNA sequence but cleave at different sites. 
Additional resources

A single-molecule counting approach for convenient and ultrasensitive measurement of restriction digest efficiencies

DNA and RNA Quantitation

Helixyte™ Green Fluorimetric dsDNA Quantitation Kit *Optimized for Broad Dynamic Range*