Thermo Fisher Scientific Components of the Reaction Mixture User guide

Brand: Thermo Fisher Scientific

Model: Components of the Reaction Mixture

Type: User guide

Protocol

Template DNA

Optimal amounts of template DNA in the 50 µl reaction

volume are in the 0.01-1 ng range for both plasmid and

phage DNA, and in the 0.1-1 µg range for genomic DNA.

Higher amounts of template increase the risk of genera-

tion of nonspecic PCR products. Lower amounts of

template reduce the accuracy of the amplication.

All routine DNA purication methods are suitable for

template preparation e.g., Genomic DNA Purication Kit

(#K0512), GeneJET™ Plasmid Miniprep Kit (#K0502).

Trace amounts of certain agents used for DNA purica-

tion, such as phenol, EDTA and proteinase K, can inhibit

thermostable DNA polymerases. Ethanol precipitation

and repeated washes of the DNA pellet with 70% ethanol

normally remove trace contaminants from DNA samples.

Primers

The recommended concentration range of primers is

0.1-1 µM. Too high primer concentrations increase the

probability of mispriming and thereby appearance of

nonspecic PCR products.

For degenerate primers and primers used for long PCR

higher primer concentrations in the range of 0.3-1 µM are

often favorable. Therefore start optimization from

standard concentrations and increase if necessary.

Mg2+ Concentration

Mg2+ in general stabilizes primer-template complexes.

PCR buffers for Taq DNA Polymerase are supplemented

with Mg2+, while in PCR with Pfu DNA Polymerase

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-specic

PCR products may appear and the PCR delity may be

reduced if the Mg2+ concentration is too high. If DNA

samples contain EDTA or other metal chelators, the Mg2+

ion concentration in the PCR mixture should be increased

accordingly (1 molecule of EDTA binds 1 molecule of

Mg2+(1)).

Recommended Mg2+ concentrations:

• Taq DNA Polymerase is supplied with two buffers:

Taq buffer with KCl and Taq buffer with (NH4)2SO4.

K+ stabilizes primer annealing whereas NH4

+ has a

destabilizing effect especially on weak hydrogen bonds

between mismatched primer-template base pairs.

Therefore for standard PCR with Taq DNA Polymerase

and 0.2 mM dNTPs the recommended MgCl2 concen-

trations are in general lower 1.5±0.25 mM when using

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

offers higher primer specicity in a broad range of

magnesium concentrations at variety of annealing

temperatures.

• For standard PCR with Pfu DNA Polymerase, 2 mM

MgSO4 is recommended. Volumes of 25 mM MgCl2 or

25 mM MgSO4 solutions required to reach a specic

concentration of magnesium ions in the 50 µl reaction

volume:

Final 1.0 1.25 1.5 1.75 2.0 2.5 3.0 4.0

Volume of 25

mM MgCl2 or

MgSO4, µl

22.5 33.5 4 5 6 8

Components of the Reaction Mixture

This protocol is for the Components of the Reaction Mixture

22 dNTPs

The recommended concentration of each dNTP is 0.2

mM. In certain PCR applications higher dNTP concentra-

tions are required. Due to the binding of Mg2+ to dNTPs,

Mg2+ concentration needs to be adjusted accordingly. It is

essential to have equal concentrations of all four nucleo-

tides (dATP, dCTP, dGTP and dTTP). If the nucleotide

concentrations are not balanced, the PCR error rate may

dramatically increase. PureExtreme® dNTP Mixes

contain either 2 mM or 10 mM, or 25 mM of each

nucleotide. The concentrations of all four dNTPs are

perfectly balanced to provide delity and to increase the

yield of PCR products.

To achieve 0.2 mM concentration of each dNTP in the

PCR mixture, use the following volumes of dNTP Mixes:

To prepare 1 ml of working solutions of dNTPs (dNTP

Mixes) from individual 100 mM dNTPs or dNTP Set, use

the following volumes of reagents:

Thermostabile DNA Polymerases

Taq DNA Polymerase. Taq DNA polymerase is the most

commonly used enzyme for PCR. It is suitable for most

amplifcation reactions that do not require high delity

enzyme or PCR products longer than 3 kb.

Normally, 1-1.5 u of Taq DNA Polymerase are recom-

mended for a 50 µl volume of a PCR mixture. Nonspecic

PCR products may appear at higher concentrations of the

polymerase. However, it may be necessary to increase the

amount of Taq DNA Polymerase to 2-3 u, if the PCR

mixture contains inhibitors, for instance, due to contami-

nation of the template DNA.

Taq DNA polymerase, if PCR is assembled at room

temperature, exhibits low but noticeable activity during

the reaction set-up. As a result, non-specic priming

events, such as mispriming or formation of primer dimers,

which occur at ambient temperatures, will lead to

generation of nonspecic amplication products during

PCR. Therefore, PCR reaction set-up should always be

performed on ice.

DreamTaq™ DNA Polymerase. DreamTaq™ DNA

Polymerase is an enhanced Taq DNA polymerase

optimized for all standard PCR applications. It ensures

higher sensitivity, longer PCR products and higher yields

compared to conventional Taq DNA Polymerase.

DreamTaq™ DNA Polymerase uses the same reaction

set-up and cycling conditions as conventional Taq DNA

Polymerase. An optimization of reaction conditions is

generally not required. It is supplied with optimized

DreamTaq™ buffer, which includes 20 mM MgCl2.

DreamTaq™ DNA Polymerase generates PCR products

with 3’-dA overhangs. PCR with DreamTaq™ DNA

Polymerase is inhibited by dUTP, but the enzyme can

incorporate modied nucleotides.

Hot Start Taq DNA Polymerases. Hot start PCR uses

enzymes, which have no activity at room temperature and

are activated only at high temperatures during PCR

cycling (e.g. TrueStart™ Hot Start Taq DNA Polymerase

or Maxima® Hot Start Taq DNA Polymerase). In hot

start PCR non-specic amplication is reduced and target

yield is increased. Using hot start DNA polymerases, PCR

can be set-up at room temperature. TrueStart™ Hot Start

Taq DNA polymerase has very short activation time (1

min) and can be used without changing of regular PCR

cycling protocol. Maxima® Hot Start Taq DNA Poly-

merase is activated in 4 min.

Pfu DNA Polymerase. Pfu DNA Polymerase is a thermo-

stable DNA polymerase with proofreading activity. It is

one of the highest delity enzymes among thermostable

DNA polymerases and is widely used in applications

which require high delity amplication, e.g. cloning and

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

delity depends on the target to be amplied and on the

presence of inhibitors in the PCR mixture. Pfu DNA

polymerase is a slower enzyme than Taq DNA polymerase

and it requires an elongation time of 2 min/kb. Also, Pfu

DNA polymerase often requires more PCR cycles to

produce sufcient amount of PCR product. Due to the

intrinsic 3’≥5’ exonuclease activity Pfu DNA polymerase

should always be the last component added to the

reaction mixture to avoid degradation of primers. It is

also recommended to use longer PCR primers. Alterna-

tively, phosphorothioate primers (exo- resistant primers)

can be used to avoid primer degradation by Pfu DNA

Polymerase (2).

PCR Enzyme Mixes. Long PCR Enzyme Mix and High

Fidelity Enzyme Mix are blends of Taq DNA Polymerase

and a thermostable DNA polymerase with a proofreading

activity. The two enzymes synergistically generate long

PCR products in greater yields and higher delity than

Taq DNA Polymerase alone. The Long PCR Enzyme Mix

is also used for efcient amplication of GC-rich DNA

regions. Normally, 1.25-2.5 u of Enzyme Mix are used in

a 50 µl volume of PCR mixture. Due to the 3’≥5’ exo-

nuclease activity of proofreading enzyme Enzyme Mixes

Volume of

PCR mixture

dNTP Mix,

2 mM each

(#R0241)

dNTP Mix,

10 mM each

(#R0191)

dNTP Mix,

25 mM each

(#R1121)

50 µl 5 µl 1 µl 0.4 µl

25 µl 2.5 µl 0.5 µl 0.2 µl

20 µl 2 µl 0.4 µl 0.16 µl

Component

dNTP Mix,

2 mM each

(#R0241)

dNTP Mix,

10 mM each

(#R0191)

dNTP Mix,

25 mM each

(#R1121)

dATP, 100 mM 20 µl 100 µl 250 µl

dTTP, 100 mM 20 µl 100 µl 250 µl

dGTP, 100 mM 20 µl 100 µl 250 µl

dCTP, 100 mM 20 µl 100 µl 250 µl

Water,

nuclease-free 920 µl 600 µl -

Total volume 1 ml 1 ml 1 ml

North America

Technical Services:

techservice.genomics@thermofisher.com

Customer Services:

customerservice.genomics@

thermofisher.com

Tel 800 235 9880

Fax 800 292 6088

Europe and Asia

Technical Services:

techservice.emea.genomics@

thermofisher.com

Customer Services:

customerservice.emea.genomics@

thermofisher.com

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Protocol

should always be last components added to the reaction

mixture to avoid degradation of primers. It is also

recommended to use longer PCR primers. Alternatively,

phosphorothioate primers (exo-resistant primers) can be

used to avoid primer degradation by enzyme mixes.

PCR Master Mixes. Thermostable DNA polymerases can

be provided in a Master Mix format, a ready to use 2X

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.

References

1. David, H., Modern Analytical Chemistry, Mc Graw

Hill, 315, 2000.

2. Skerra, A., Phosphorothioate primers improve

the amplication of DNA sequences by DNA

polymerases with proofreading activity, Nucleic

Acids Res., 20, 3551-3554, 1992.

Thermo Fisher Scientific Components of the Reaction Mixture User guide

Brand: Thermo Fisher Scientific

Model: Components of the Reaction Mixture

Type: User guide

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Thermo Fisher Scientific Components of the Reaction Mixture User guide

Thermo Fisher Scientific Components of the Reaction Mixture User guide

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