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Restriction Enzyme Digestion • Add reagents in following order: x uL dH20 (enough to bring reaction volume to 50 uL) y uL of DNA (depends on concentration) 5 uL of appropriate 10X buffer 1 5 uL of 10X BSA (even if not required) 1 z uL Restriction Enzyme(s) (keep on ice!) 2,3 • Tap the reaction tube gently several times to mix • Pulse spin the reaction to the bottom of the tube • Incubate the reaction at 37°C 4 NOTES: 1= Many NEB enzymes now work in the new buffer system called CutSmart. CutSmart is basically NEB Buffer #4 and BSA combined (10X solution). Before using CutSmart, ensure your enzyme’s compatability on www.neb.com 2= Restriction enzyme activity is measured in “units.” One unit is defined as the amount of the enzyme required to digest 1 ug of DNA in 60 minutes. 10-fold overdigestion is recommended. In our lab, use 10 units of enzyme for DNA amounts of 1 ug or less. Add 10 units for each additional 0.1-1 ug of DNA being digested (e.g. for 3.5 ug of DNA, use 40 units of enzyme) 3= The volume of restriction enzyme used in a reaction should not exceed 10% of the total reaction volume to reduce star activity 4= For standard diagnostic digestions, a 1-2 hour digestion should be sufficient. For digestions whose product will be used for subsequent cloning steps, an overnight (16 hour) digestion is recommended