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Focus: PCR
Performance Advantages Designed into Promega's PCR Master Mix
Promega's
PCR Master Mix is designed for the rapid and convenient
amplification of many common genomic and cDNA templates. PCR Master Mix has been optimized
to produce PCR products up to 2kb in size and it exhibits superior stability. PCR Master
Mix is provided as a 2X solution that contains all the necessary reaction components
except primers and template DNA, thus greatly reducing setup time and pipetting steps. |
By Mark Denhart, M.S. and Vinayak Doraiswamy,
Ph.D.
Promega Corporation
Introduction
PCR amplification(a) is routinely used in molecular
biology. Typically, the common reaction components (e.g., buffer, dNTPs, MgCl2
and DNA polymerase) are mixed together to create a "master mix", which is then
aliquoted to individual reaction tubes. Primers and template DNA are added just prior to
performing PCR. Promega's new PCR Master Mix(a,b) is truly a master mix solution that contains
all the reaction components required at 2X concentration for amplification. One need only
add reaction-specific primers, DNA template and the provided Nuclease-Free Water to
achieve 1X working concentration.
Sensitivity
Increasingly, scientists are required to analyze minute amounts of DNA, and thus PCR
amplification sensitivity has become critical. To demonstrate the sensitivity of
amplifications performed with PCR Master Mix, we amplified a fragment of the a1-antitrypsin gene from serial dilutions of Human Genomic DNA (Cat.# G3041). a1-antitrypsin is a single copy per genome gene. As evident in Figure 1, we were able to amplify a
360bp fragment from dilutions containing as few as two copies per reaction of this gene
using PCR Master Mix.
Template Versatility
DNA templates come from species as varied as bacteria, plants and humans. As seen in Figure 2, we tested PCR Master Mix
using serial dilutions of DNA from many such sources including bacteria, lambda phage,
soybean, human and viral ssRNA (as a template to amplify a fragment of the 5´-UTR).
Stability
We used two approaches to test the stability of PCR Master Mix: i) time in storage and
ii) number of freeze-thaw events. First, PCR Master Mix was thawed at room temperature and
then stored at 4°C for up to 15 months. PCR amplification of a 360bp fragment of the
human a1-antitrypsin gene was performed following storage. In Figure 3, PCR Master Mix amplified the
target with no noticeable changes in PCR quality even after 15 months in storage.
Second, PCR Master Mix was thawed and refrozen up to 10 times before use. Two rates of
freeze-thaw events were performed, "slow" and "fast" thaws. For the
slow thaw, PCR Master Mix was removed from storage at –20°C and allowed to thaw at
room temperature (~25°C); for the fast thaw, it was removed from –70°C and placed
directly in a 50°C water bath until thawed. PCR amplification was performed after each
thaw event had been repeated five or 10 times. As evident in Figure 4, there was no noticeable drop
in amplification product after 10 freeze-thaw cycles.
Robustness
Thermal cycling instruments may exhibit a temperature deviation of 1-2 degrees. PCR
Master Mix contains a specialized buffer to keep the Taq DNA Polymerase(a) stable at elevated temperatures. This can be especially important
in cases when the amplification profile requires an elevated denaturation temperature or
an increased number of cycles (3).
We tested the tolerance of PCR Master Mix at three elevated denaturation
temperatures—95, 97 and 99°C—by amplifying a 360bp fragment of the a1-antitrypsin gene from Human Genomic DNA (Cat.# G3041). In Figure 5, Promega PCR Master Mix was
compared to another commercially available PCR mix under the same conditions.
Supplier L's PCR master mix produced no detectable product at denaturation temperatures
>95°C. Promega's PCR Master Mix amplified product with equal efficiency at all
denaturation temperatures.
Scalability
Finally, we assessed scalability in terms of reaction volume. Different applications
require PCR to be performed in different volumes, typically in the range of 10 to 50µl.
PCR Master Mix produces consistent amplification results when used in reactions with final
volumes between 10 and 50µl (Figure 6).
Conclusions
In this article, we show that the PCR Master Mix offers improved sensitivity,
amplifying a target sequence from as few as two copies of starting template. PCR Master
Mix was shown to effectively amplify sequences in sizes ranging from 125bp to 1.3kb. PCR
Master Mix amplified DNA isolated from mammalian, plant, bacterial and viral sources with
equal success. In addition, stability is a key feature of PCR Master Mix. We describe here
experiments that show that the Master Mix is fully scalable and sensitive enough to detect
low copy number templates and is also stable when stored for long periods at 4°C even
after multiple freeze-thaw cycles. In addition, Nuclease-Free Water is included in a
convenient separate vial to ensure high-quality results.
As PCR amplification has become central to molecular biology research, methods to
streamline reaction setup are required. Promega's PCR Master Mix, formulated as a 2X
solution, offers single-tube format for reaction setup, reducing pipetting times, steps
and errors as well as greatly reducing reagent waste.
References
- Ilag, L.L. et al. (1991) The Escherichia coli hemL
gene encodes glutamate 1-semialdehyde aminotransferase. J. Bacteriol. 173,
3408-3413.
- Lin, J.-J. et al. (2000) Detection of plant genes using a rapid,
nonorganic DNA purification method. BioTechniques 28,
346-350.
- Ruano, G. et al. (1992) Heat-soaked PCR: an efficient method for
DNA amplification with applications to forensic analysis. BioTechniques 13,
266-274.
(a)The PCR process is covered by patents issued and
applicable in certain countries. Promega does not encourage or support the unauthorized or
unlicensed use of the PCR process.
(b)For Laboratory Use.
*Products may be covered by pending or issued patents. Please visit
our patent and trademark web page for more information.
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Figure
1. Detection of low copy number templates using PCR Master Mix. |
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Figure
2. PCR amplification using DNA from different sources. |
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Figure
3. Stability of PCR Master Mix at 4°C. |
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Figure
4. Stability of PCR Master Mix through multiple freeze-thaw events. |
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Figure
5. Stability of PCR Master Mixes at high denaturation temperatures. |
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Figure
6. Scalability of the PCR Master Mix. |
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