The nuclease test will vary in components depending on the product whether it is the enzyme or PCR master mix. The enzyme buffer is not added when testing the PCR Master Mix.
The incubation times may vary depending on the product being tested. We typically use 4 hours for enzymes and 16 to 20 hours for other products( DNA loading dye ).
Summarily, the nuclease test involves incubation of a reaction mixture containing a minimum of 20 units of enzyme (test enzyme/ PCR master mix) with 1 μg of pOBL1 or PUC19 plasmid DNA-50 pg/uL for 4 hours at 37°C.
The nuclease activity of the test product is determined by visualizing the migration pattern of the DNA plasmid on 1.5% (w/v) agarose gel electrophoresis.
In our lab we use pOBL1 plasmid but any circular double stranded DNA can be used.