Polymerases Quality Control
New England Biolabs is committed to identifying, cloning and characterizing a variety of DNA polymerases suitable for current and future research applications. Whether mesophilic or thermophilic, holoenzymes, proteolytic derivatives or forms with altered exonuclease or nucleotide insertion properties, we strive to provide the diversity essential to enable and optimize a wide array of protocols.
Quality Controls
DNA polymerases undergo extensive quality control testing at New England Biolabs. There are two basic
types of tests; those designed to monitor specific DNA polymerase function, and those designed to detect
any contaminating enzyme species that might interfere with DNA polymerase function.
To ensure consistency between individual lots of purified DNA polymerases, determinations of activity levels are performed on defined, characterized, DNA substrates. Each lot is routinely reassayed to ensure that full activity is retained under recommended storage conditions.
Assay for Nonspecific Endonucleases
To assay for nonspecific endonuclease contamination, polymerase preparations are incubated with supercoiled
fx174 (RF I) DNA and the production of nicked DNA (RF II) is monitored. Since a single nonspecific
nick converts RF I to RF II, production of RF II measures the presence of endonucleases. Aliquots are
incubated with 1 µg of RF I DNA in a reaction volume of 50 µl using the recommended reaction
buffer. Incubations are performed for 4 hours at the recommended temperature (thermophilic
DNA polymerases prepared from recombinant sources are also tested at 37°C). 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.
Assay for Nuclease Contamination
All polymerases are incubated for at least 4 hours 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 (with dNTPs present if the polymerase has a 3´-> 5´ exonuclease
activity that requires the presence of dNTPs for proper regulation). 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 leave the substrate unaltered
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 exonuclease deficient polymerases 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 at the recommended temperature (thermophilic DNA polymerases prepared
from recombinant sources are also tested at 37°C) for 4 hours. 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.