With the introduction of the Pwo Master, Roche Applied
Science now offers a new, ready-to-use proofreading
master mix for the robust amplification of fragments up to
3 kb, providing the highest possible fidelity during PCR
Pwo Master is a premixed double-concentrated formulation
that contains PwoSUPER YIELD DNA Polymerase,
a new optimized buffer system, and PCR-Grade
Nucleotides. After simply adding the template DNA and
the primers and adjusting the final volume to 50 l with
PCR-Grade Water (provided with the Pwo Master), the
reaction can be started.
The reaction setup of a large number of samples for PCR
analysis requires a multitude of pipetting and handling
steps. Every step in PCR is critical to the quality of the final
result; pipetting and handling errors, as well as assay contamination,
can negatively impact the experimental outcome,
leading to false-negative or false-positive results.
As a result, PCRs must often be repeated, resulting in
wasted effort and reagent expense. The availability of master mixes simplifies the PCR setup
and circumvents many problems.
Enzymology of Pwo Master
The core component of the Pwo Master is the proofreading
polymerase Pwo DNA Polymerase. Pwo DNA
Polymerase is a highly processive 5′3′ DNA polymerase
that possesses 3′5′ exonuclease activity, also known as proofreading activity. This proofreading activity
results in an approximate 18-fold increase in the
fidelity of DNA synthesis compared with Taq DNA polymerase.
PwoSUPER YIELD DNA Polymerase combines Pwo DNA
Polymerase with a new optimized buffer system which
enhances the enzy
matic properties of the enzyme, resulting
in higher yields and additional robustness of the
amplification reaction without changing the fidelity of
Advantages of Pwo Master
Avoid the need to determine the optimal buffer
conditions for your proofreading PCRs
Improve the reproducibility and consistency of
your proofreading PCRs
- The Mg2+ concentration of the master mix in combination
with the specially formulated buffer system
allows the amplification of most fragments up to 3 kb
with high yield and specificity (Figure 1), and the high
fidelity you expect from a proofreading polymerase
- There is no need to titrate the optimal ionic conditions
for a given assay or to perform cumbersome adjustments
of the reagent compositions (Figure 1).
Save time the Pwo Master is ready when you are
- The number of pipetting steps during PCR setup is
- The risk of contaminating the reaction mix during PCR
setup is reduced.
- The analysis of your results is straightforward.
Contents of Pwo Master
- The Pwo Master is stored at 2 8C and can be used
immediately; no freezing or thawing is necessary.
β-Glucuronidase from E. coli K12
New improved quality!
- Pwo Master: 10 vials of double-concentrated master
mix, 10 reactions per vial. Each vial contains 25 units
PwoSUPER YIELD DNA Polymerase, reaction buffer with
4mM MgCl2 and PCR-Grade Nucleotides (dATP, dCTP,
dGTP, dTTP, each 0.4 mM) in a total volume of 250 l.
- PCR-Grade Water: four vi
als of 1 ml each.
Roche Applied Science recently introduced an improved
β-Glucuronidase (GRD) formulation which replaces the
previous product. The new quality exhibits higher purity
and specific activity compared with products previously
on the market (Figure).
GRD hydrolyses β-glucuronic acid (β-GlcA) esters
(β-glucuronosides) of a variety of compounds, including
steroids, aryl alcohols, drugs, and metabolites. GRD also
hydrolyses polysaccharides that contain β-GlcA linked to
other sugars. GRD is highly specific for the carbohydrate
portion of the molecule but has very little specificity for
the molecule conjugated to β-GlcA. The main research
- Analysis of steroid conjugates
- Doping analysis (horses)
- Drug and alcohol testing
- Tests in pathology labs
The performance in steroid analysis and doping analysis
was demonstrated by Ute Mareck, German Sport
University, Cologne, Germany, and Thierry Rousseau, National Doping Testing Laboratory (LNDD), Chtenay-
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