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P rotocol

Components of the Reaction Mixture

This protocol is for the Components of the Reaction Mixture

Template DNA samples contain EDTA or other metal chelators, the Mg2+
Optimal amounts of template DNA in the 50 µl reaction ion concentration in the PCR mixture should be increased
volume are in the 0.01-1 ng range for both plasmid and accordingly (1 molecule of EDTA binds 1 molecule of
phage DNA, and in the 0.1-1 µg range for genomic DNA. Mg2+(1)).
Higher amounts of template increase the risk of genera-
tion of nonspecific PCR products. Lower amounts of Recommended Mg 2+ concentrations:
template reduce the accuracy of the amplification. • Taq DNA Polymerase is supplied with two buffers:
All routine DNA purification methods are suitable for Taq buffer with KCl and Taq buffer with (NH4)2SO4.
template preparation e.g., Genomic DNA Purification Kit K+ stabilizes primer annealing whereas NH4+ has a
(#K0512), GeneJET™ Plasmid Miniprep Kit (#K0502). destabilizing effect especially on weak hydrogen bonds
Trace amounts of certain agents used for DNA purifica- between mismatched primer-template base pairs.
tion, such as phenol, EDTA and proteinase K, can inhibit Therefore for standard PCR with Taq DNA Polymerase
thermostable DNA polymerases. Ethanol precipitation and 0.2 mM dNTPs the recommended MgCl2 concen-
and repeated washes of the DNA pellet with 70% ethanol trations are in general lower 1.5±0.25 mM when using
normally remove trace contaminants from DNA samples. Taq buffer with KCl compared to 2.0±0.5 mM when
using Taq buffer with (NH4)2SO4. Due to antagonistic
effects of NH4+ and Mg2+, Taq buffer with (NH4)2SO4
Primers
offers higher primer specificity in a broad range of
The recommended concentration range of primers is magnesium concentrations at variety of annealing
0.1-1 µM. Too high primer concentrations increase the temperatures.
probability of mispriming and thereby appearance of
• For standard PCR with Pfu DNA Polymerase, 2 mM
nonspecific PCR products.
MgSO4 is recommended. Volumes of 25 mM MgCl2 or
For degenerate primers and primers used for long PCR 25 mM MgSO4 solutions required to reach a specific
higher primer concentrations in the range of 0.3-1 µM are concentration of magnesium ions in the 50 µl reaction
often favorable. Therefore start optimization from volume:
standard concentrations and increase if necessary.

Final 1.0 1.25 1.5 1.75 2.0 2.5 3.0 4.0


Mg 2+ Concentration
Mg2+ in general stabilizes primer-template complexes.
Volume of 25
PCR buffers for Taq DNA Polymerase are supplemented
mM MgCl2 or 2 2.5 3 3.5 4 5 6 8
with Mg2+, while in PCR with Pfu DNA Polymerase
MgSO4, µl
MgSO4 is a preferable component. Due to the binding of
Mg2+ to dNTPs, primers and DNA templates, Mg2+
concentration needs to be optimized for maximal PCR
yield. The recommended concentration range is 1-4 mM.
If the Mg2+ concentration is too low, the yield of PCR
product could be reduced. On the contrary, non-specific
PCR products may appear and the PCR fidelity may be
reduced if the Mg2+ concentration is too high. If DNA
22 dNTPs PCR. Therefore, PCR reaction set-up should always be
The recommended concentration of each dNTP is 0.2 performed on ice.
mM. In certain PCR applications higher dNTP concentra- DreamTaq™ DNA Polymerase. DreamTaq™ DNA
tions are required. Due to the binding of Mg2+ to dNTPs, Polymerase is an enhanced Taq DNA polymerase
Mg2+ concentration needs to be adjusted accordingly. It is optimized for all standard PCR applications. It ensures
essential to have equal concentrations of all four nucleo- higher sensitivity, longer PCR products and higher yields
tides (dATP, dCTP, dGTP and dTTP). If the nucleotide compared to conventional Taq DNA Polymerase.
concentrations are not balanced, the PCR error rate may DreamTaq™ DNA Polymerase uses the same reaction
dramatically increase. PureExtreme® dNTP Mixes set-up and cycling conditions as conventional Taq DNA
contain either 2 mM or 10 mM, or 25 mM of each Polymerase. An optimization of reaction conditions is
nucleotide. The concentrations of all four dNTPs are generally not required. It is supplied with optimized
perfectly balanced to provide fidelity and to increase the DreamTaq™ buffer, which includes 20 mM MgCl2.
yield of PCR products. DreamTaq™ DNA Polymerase generates PCR products
with 3’-dA overhangs. PCR with DreamTaq™ DNA
dNTP Mix, dNTP Mix, dNTP Mix,
Volume of 2 mM each Polymerase is inhibited by dUTP, but the enzyme can
10 mM each 25 mM each
PCR mixture incorporate modified nucleotides.
(#R0241) (#R0191) (#R1121)
50 µl 5 µl 1 µl 0.4 µl Hot Start Taq DNA Polymerases. Hot start PCR uses
25 µl 2.5 µl 0.5 µl 0.2 µl enzymes, which have no activity at room temperature and
are activated only at high temperatures during PCR
20 µl 2 µl 0.4 µl 0.16 µl
cycling (e.g. TrueStart™ Hot Start Taq DNA Polymerase
or Maxima® Hot Start Taq DNA Polymerase). In hot
To achieve 0.2 mM concentration of each dNTP in the start PCR non-specific amplification is reduced and target
PCR mixture, use the following volumes of dNTP Mixes: yield is increased. Using hot start DNA polymerases, PCR
can be set-up at room temperature. TrueStart™ Hot Start
dNTP Mix, dNTP Mix, dNTP Mix, Taq DNA polymerase has very short activation time (1
Component 2 mM each 10 mM each 25 mM each min) and can be used without changing of regular PCR
(#R0241) (#R0191) (#R1121) cycling protocol. Maxima® Hot Start Taq DNA Poly-
dATP, 100 mM 20 µl 100 µl 250 µl merase is activated in 4 min.
dTTP, 100 mM 20 µl 100 µl 250 µl Pfu DNA Polymerase. Pfu DNA Polymerase is a thermo-
dGTP, 100 mM 20 µl 100 µl 250 µl stable DNA polymerase with proofreading activity. It is
dCTP, 100 mM 20 µl 100 µl 250 µl one of the highest fidelity enzymes among thermostable
Water, DNA polymerases and is widely used in applications
920 µl 600 µl - which require high fidelity amplification, e.g. cloning and
nuclease-free
Total volume 1 ml 1 ml 1 ml expression. Normally, 1.25-2.5 u of Pfu DNA Polymerase
are used in a 50 µl volume of PCR mixture. The actual
amount of enzyme required for optimal PCR yield and
To prepare 1 ml of working solutions of dNTPs (dNTP fidelity depends on the target to be amplified and on the
Mixes) from individual 100 mM dNTPs or dNTP Set, use presence of inhibitors in the PCR mixture. Pfu DNA
the following volumes of reagents: polymerase is a slower enzyme than Taq DNA polymerase
and it requires an elongation time of 2 min/kb. Also, Pfu
Thermostabile DNA Polymerases DNA polymerase often requires more PCR cycles to
produce sufficient amount of PCR product. Due to the
Taq DNA Polymerase. Taq DNA polymerase is the most
intrinsic 3’≥5’ exonuclease activity Pfu DNA polymerase
commonly used enzyme for PCR. It is suitable for most
should always be the last component added to the
amplifcation reactions that do not require high fidelity
reaction mixture to avoid degradation of primers. It is
enzyme or PCR products longer than 3 kb.
also recommended to use longer PCR primers. Alterna-
Normally, 1-1.5 u of Taq DNA Polymerase are recom- tively, phosphorothioate primers (exo- resistant primers)
mended for a 50 µl volume of a PCR mixture. Nonspecific can be used to avoid primer degradation by Pfu DNA
PCR products may appear at higher concentrations of the Polymerase (2).
polymerase. However, it may be necessary to increase the
PCR Enzyme Mixes. Long PCR Enzyme Mix and High
amount of Taq DNA Polymerase to 2-3 u, if the PCR
Fidelity Enzyme Mix are blends of Taq DNA Polymerase
mixture contains inhibitors, for instance, due to contami-
and a thermostable DNA polymerase with a proofreading
nation of the template DNA.
activity. The two enzymes synergistically generate long
Taq DNA polymerase, if PCR is assembled at room PCR products in greater yields and higher fidelity than
temperature, exhibits low but noticeable activity during Taq DNA Polymerase alone. The Long PCR Enzyme Mix
the reaction set-up. As a result, non-specific priming is also used for efficient amplification of GC-rich DNA
events, such as mispriming or formation of primer dimers, regions. Normally, 1.25-2.5 u of Enzyme Mix are used in
which occur at ambient temperatures, will lead to a 50 µl volume of PCR mixture. Due to the 3’≥5’ exo-
generation of nonspecific amplification products during nuclease activity of proofreading enzyme Enzyme Mixes
should always be last components added to the reaction References
mixture to avoid degradation of primers. It is also 1. David, H., Modern Analytical Chemistry, Mc Graw
recommended to use longer PCR primers. Alternatively, Hill, 315, 2000.
phosphorothioate primers (exo-resistant primers) can be 2. Skerra, A., Phosphorothioate primers improve
used to avoid primer degradation by enzyme mixes. the amplification of DNA sequences by DNA

P rotocol
PCR Master Mixes. Thermostable DNA polymerases can polymerases with proofreading activity, Nucleic
be provided in a Master Mix format, a ready to use 2X Acids Res., 20, 3551-3554, 1992.
concentrated solution, which includes DNA polymerase
together with a PCR buffer and nucleotides. The Master
mix is the most convenient and cost effective product for
routine or high throughput PCR, where time for setting
up a reaction and reproducibility of results are most
important factors.

We offer two PCR Master Mixes. The PCR Master Mix


(2X) contains Taq DNA polymerase and is suitable for
routine PCR. The PyroStart™ Fast PCR Master Mix (2X)
contains a hot start Taq DNA polymerase and is formu-
lated to work in fast thermal cycling conditions to reduce
time not only dedicated to PCR set-up, but also to PCR
cycling. PCR of less than 1 kb target can be completed in
25 min using this product.

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