DNA/RNA Modifying Enzymes Quality Controls
Assay for Nonspecific Endonucleases
To assay for nonspecific endonuclease contamina-tion, modifying
enzymes are incubated with a supercoiled plasmid substrate. A single nonspecific nick in the RF I DNA converts
it to the RF II form (nicked circle). Aliquots are incubated with 1 µg of RF I (supercoiled form) DNA
in a reaction volume of 50 µl using the recommended reaction buffer. Incubations are performed for 4 hours
at the recommended temperature. The two forms are easily distinguished on agarose gels and the percent conversion
from RF I to RF II is reported on the Technical Data Card provided with each enzyme.
Overnight Assay for Nuclease Contamination
All modifying enzymes are incubated overnight in their recommended
reaction buffer with 1 µg of substrate DNA (Hind III fragments of Lambda DNA or Hae III fragments
of fx174 DNA) in a volume of 50 µl. The characteristic banding pattern of the DNA substrate 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
DNases. The maximum number of units that can be incubated overnight is reported on the Technical Data Card provided
with each enzyme. Enzymes used for RNA work are tested for ribonuclease contamination by incubation with 5 µg
of MS2 RNA in the supplied reaction buffer for 1 hour at 37°C, followed by denaturing agarose gel electrophoresis.
Assay for Exonuclease Contamination
All modifying enzymes are incubated with 1 µg of a mixture of single
and double-stranded, 3H-labeled E. coli DNA (200,000 cpm/µg) in a reaction volume of 50 µl. The
supplied reaction buffer is used for this test. Incubations are performed for 4 hours at the recommended temperature.
Exonuclease contamination is indicated by the percent of the total labeled DNA in the reaction that
has been rendered TCA-soluble. The limit of detectability of this assay is approximately 0.05%.
The results of this assay are given on the Technical Data Card provided with each enzyme.
Assay for Phosphatase Contamination
Phosphatase contamination is revealed by the loss of radioactivity from
a 5´ 32P-labeled eight base deoxyribonucleotide d(T)8, as determined by denaturing polyacrylamide
gel electrophoresis and autoradiography. Preparations of enzyme are incubated for 4 hours under standard assay
conditions in 50 µl reactions containing this substrate.
Methylase Quality Assurance
All methylases are assayed for the presence of contaminating endonucleases
and exonucleases in a low salt, MgCl2 containing buffer with no EDTA. This buffer is used because EDTA or high
salt concentrations inhibit contaminating activities.