Quality Control Assays-Maximum Units per Reaction When using procedures requiring larger quantities of enzyme and/or extended reaction times, an appreciation of the quality control data can help determine a safe amount of enzyme for your Overnight Assay Increasing amounts of restriction endonuclease are incubated overnight(typically for 16 hours)in their recommended buffer with l ug of substrate dNa in a volume of 50 ul. The characteris- tic limit digest banding pattern produced by the enzyme in one hour is compared to the pattern produced from an excess of enzyme incubated overnight. A sharp, unaltered pattern under these conditions is an indication that the enzyme preparation is free of detectable levels of nonspecific endonucleases. The maximum number of units yielding an unaltered pattern is eported. Enzymes listing 100 units or more, a 1600-fold over digestion(100 units x 16h), will not degrade DNA up to megabase size in mapping experiments and can be assumed to be virtually free of nonspecific endonuclease (Davis, T. and Robinson, D unpublished observations Nicking Assay Another sensitive test for contaminating endonucleases is a four hour incubation with a supercoiled plasmid that lacks a site for the enzyme being tested. The supercoil is very sensi- tive to nonspecific nicking by a single-stranded endonuclease, cleavage by a double-stranded endonuclease, or topoisomerase activity. If a single-stranded nick occurs, the supercoiled mole- cule. RFl. unwinds and assumes the circular form rFil. If a double-stranded cleavage occurs, the circle will become linear High levels of single-stranded nicking leads to linear DNA All three forms of DNa have distinct electrophoretic mobilities on agarose gels. Enzymes converting 5%or less of the plasmid o relaxed form using 100 units of enzyme for four hours can be considered virtually free of nicking activity. High-salt buffers, especially at elevated temperature, can cause some conversion to relaxed form. A control reaction, including buffer and dnA but lacking enzyme, is incubated and run on the agarose gel fo Exonuclease Assay Suppliers use a variety of assays to check for exonuclease activ ity. a general assay mixture contains a restriction endonuclease 34 Robinson et alQuality Control Assays—Maximum Units per Reaction When using procedures requiring larger quantities of enzyme and/or extended reaction times, an appreciation of the quality control data can help determine a safe amount of enzyme for your application. Overnight Assay Increasing amounts of restriction endonuclease are incubated overnight (typically for 16 hours) in their recommended buffer with 1mg of substrate DNA in a volume of 50ml. The characteris￾tic limit digest banding pattern produced by the enzyme in one hour is compared to the pattern produced from an excess of enzyme incubated overnight. A sharp, unaltered pattern under these conditions is an indication that the enzyme preparation is free of detectable levels of nonspecific endonucleases. The maximum number of units yielding an unaltered pattern is reported. Enzymes listing 100 units or more, a 1600-fold over digestion (100 units ¥ 16h), will not degrade DNA up to megabase size in mapping experiments and can be assumed to be virtually free of nonspecific endonuclease (Davis, T. and Robinson, D., unpublished observations). Nicking Assay Another sensitive test for contaminating endonucleases is a four hour incubation with a supercoiled plasmid that lacks a site for the enzyme being tested. The supercoil is very sensi￾tive to nonspecific nicking by a single-stranded endonuclease, cleavage by a double-stranded endonuclease, or topoisomerase activity. If a single-stranded nick occurs, the supercoiled mole￾cule, RFI, unwinds and assumes the circular form, RFII. If a double-stranded cleavage occurs, the circle will become linear. High levels of single-stranded nicking leads to linear DNA. All three forms of DNA have distinct electrophoretic mobilities on agarose gels. Enzymes converting 5% or less of the plasmid to relaxed form using 100 units of enzyme for four hours can be considered virtually free of nicking activity. High-salt buffers, especially at elevated temperature, can cause some conversion to relaxed form. A control reaction, including buffer and DNA but lacking enzyme, is incubated and run on the agarose gel for comparison. Exonuclease Assay Suppliers use a variety of assays to check for exonuclease activ￾ity. A general assay mixture contains a restriction endonuclease 234 Robinson et al