Реактивы для ПЦР

1. Thermus aquaticus DNA Polymerase (кат. № PTT-01)

Description: Taq DNA Polymerase (recombinant form of the enzyme from the thermophilic eubacterium Thermus aquaticus strain YT-1) consists of a single polypeptide chain with a molecular weight 94 kDa. It is a highly processive 5'-3' DNA polymerase lacking 3'-5' exonuclease activity. The enzyme is highly purified and is free of nonspecific endo- or exonucleases. This Taq DNA polymerase has a nontemplate-dependent activity which adds a single deoxyadenosine (A) to the 3' ends of PCR products (3'-A overhangs). This property allows easily and efficiently ligating the PCR products in TA cloning vectors.

Unit Definition: One unit of activity is the amount of enzyme required to incorporate 10 nmoles of dNTP into acid-insoluble material in 30 minutes at 72^oC.

Concentration: 5 u/ml.

Storage (Dilution) Buffer: 25 mM Tris-HCl, pH8.0, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol, 0.75% Triton X-100. Store at -20^oC.

Reaction Buffer (x10): 670 mM Tris-HCl, pH 8.8, 166 mM (NH₄)₂SO₄, 0.1% Tween 20, 25 mM MgCl_2.

It is possible to use other buffers for Taq polymerase.

Application: PCR, DNA labeling

Quality Control Tests: Activity, SDS-PAGE purity, absence of endonucleases/nickases and exonucleases.

2. Thermus aquaticus DNA Polymerase, DNA E. coli minus (кат. № PTT-02)

Description: Taq DNA Polymerase, DNA E. coli minus is a recombinant form of Thermus aquaticus DNA Polymerase, highly purified through a proprietary separation process to ensure that contaminating bacterial DNA sequences are substantially reduced. This purification ensured that non-target, false positive PCR products will be effectively minimized when amplifying bacterial sequences. This product is especially useful for low copy number PCR amplifications. The enzyme is tested to verify that less than 10 copies of bacterial 16S ribosomal RNA gene sequences are present in a standard 2 Unit aliquot.

Unit Definition: One unit of activity is the amount of enzyme required to incorporate 10 nmoles of dNTP into acid-insoluble material in 30 minutes at 72^oC.

Concentration: 5 u/ml.

Storage (Dilution) Buffer: 25 mM Tris-HCl, pH8.0, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol, 0.75% Triton X-100. Store at -20^oC.

Reaction Buffer (x10) (Mg²⁺ Minus): 670 mM Tris-HCl, pH 8.8, 166 mM (NH₄)₂SO₄, 0.1% Tween 20.

Optimal concentration of MgCl₂ is 2.5 mM. It is possible to use other buffers for Taq polymerase.

Application: PCR, PCR diagnostic of Enterobacteriae.

Quality Control Tests: Activity, SDS-PAGE purity, absence of endonucleases/nickases and exonucleases

3. Thermostar DNA Polymerase (кат. № PTT-05)

Description: Thermostar DNA polymerase is an ultra-pure thermostable, recombinant enzyme designed to improve PCR process by providing highly specific amplification conditions. Use of Thermostar DNA polymerase prevents nonspecific primer annealing and the formation of primer-dimers. Thermostar DNA polymerase is supplied in an inactive state with no polymerase activity. It is activated by a 15-minute incubation at 95°C.

Attention! The enzyme has no activity in PCR buffers with pH 8.8 - 9.0. We strongly recommend to use buffers with pH 8.3. The activation conditions (15-minute incubation at 95°C) are extremely important. Great attention should be paid to the actual temperature (95°C) inside tube.

Unit Definition: One unit of activity is the amount of enzyme required to incorporate 10 nmoles of dNTP into acid-insoluble material in 30 minutes at 72^oC.

Concentration: 5 u/ml.

Storage (Dilution) Buffer: 20 mM Tris-HCl, pH 8.0, 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5% NP 40, 0.5% Tween 20, 50% glycerol. Store at -20^oC.

Reaction Buffer (10x): 100 mM Tris-HCl, pH 8.3, 500 mM KCl, 25 mM MgCl₂.

Application: PCR with high specificity

Quality Control Tests: Activity, SDS-PAGE purity, absence of endonucleases/nickases and exonucleases.

4. Hot-rescue DNA Polymerase (кат. № PTT-8)

Description: Hot-rescue DNA Polymerase is recombinant Taq DNA Polymerase complexed with proprietary antibody that inhibits polymerase activity. Due to specific binding of the inhibitor Hot-rescue DNA Polymerase is provided in an inactive form. Antibody provides an automatic “hot start” for Taq DNA Polymerase in PCR. Hot-rescue DNA Polymerase is activated by 10-minute incubation at 95°C.

Unit Definition: One unit of activity is the amount of enzyme required to incorporate 10 nmoles of dNTP into acid-insoluble material in 30 minutes at 72^oC.

Concentration: 5 u/ml.

Storage (Dilution) Buffer: 25 mM Tris-HCl, pH8.0, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol, 0.75% Triton X-100. Store at -20^oC.

Reaction Buffer (x10): 670 mM Tris-HCl, pH 8.8, 166 mM (NH₄)₂SO₄, 0.1% Tween 20, MgCl₂ minus.

Solution of 25 mM MgCl₂ is included.

Optimal concentration of MgCl₂ is more than 1,5 mM.

It is possible to use other buffers for Taq polymerase.

Application: PCR with high specificity, real time PCR

Quality Control Tests: Activity, SDS-PAGE purity, absence of endonucleases/nickases and exonucleases.

5. Seq pol (кат. № PTT-09)

Description: Seq pol (recombinant modified form of the enzyme from the thermophilic eubacterium Thermus aquaticus strain YT-1). This enzyme preparation contains very low 5'-3' exonuclease activity and incorporates dideoxynucleotides more efficiently as compared with wild type of Taq DNA Polymerase.

Unit Definition: One unit of activity is the amount of enzyme required to incorporate 10 nmoles of dNTP into acid-insoluble material in 30 minutes at 72^oC.

Concentration: 5 u/ml.

Storage (Dilution) Buffer: 25 mM Tris-HCl, pH8.0, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol, 0.75% Triton X-100. Store at -20^oC.

Reaction Buffer (x10) : 670 mM Tris-HCl, pH 8.8, 166 mM (NH₄)₂SO₄, 0.1% Tween 20, 25 mM MgCl₂

Application: Sequencing

Quality Control Tests: Activity, SDS-PAGE purity, absence of endonucleases/nickases and exonucleases.

6. ON-Taq DNA Polymerase (кат. № PTT-10)

Description: ON-Taq DNA Polymerase is recombinant Taq DNA Polymerase complexed with Hot Start Compound that inhibits polymerase activity. Due to specific binding of the inhibitor ON-Taq DNA Polymerase is provided in an inactive form Hot Start Compound provides an automatic “hot start” for Taq DNA Polymerase in PCR. ON-Taq DNA Polymerase is activated at rise in temperature above 45°C.

Unit Definition: One unit of activity is the amount of enzyme required to incorporate 10 nmoles of dNTP into acid-insoluble material in 30 minutes at 72^oC.

Concentration: 5 u/ml.

Storage (Dilution) Buffer: 25 mM Tris-HCl, pH8.0, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol, 0.75% Triton X-100. Store at -20^oC.

Reaction Buffer (x10): 670 mM Tris-HCl, pH 8.8, 166 mM (NH₄)₂SO₄, 0.1% Tween 20, 25 mM MgCl₂.

It is possible to use other buffers for Taq polymerase.

Application: PCR with high specificity

Quality Control Tests: Activity, SDS-PAGE purity, absence of endonucleases/nickases and exonucleases.

7. M-MLV Reverse Transcriptase (M-MLV RT) (кат. № PMM-01)

Description: M-MLV Reverse Transcriptase (M-MLV RT) (recombinant modified form of the Reverse Transcriptase from the Moloney Murine Leukemia Virus) is an RNA-dependent DNA polymerase. The enzyme is a product of a pol gene of M-MLV and consists of a single subunit with a molecular weight of 71 kDa. M-MLV RT has a low intrinsic RNase H activity.

Unit Definition: One unit of activity is the amount of enzyme required to incorporate 1 nmoles of dNTP into acid-precipitable material in 10 minutes at 37^oC using poly(rA)-oligo(dT)₅₀ as template-primer.

Concentration: 50 u/ml.

Storage (Dilution) Buffer: 50 mM Tris-HCl, pH8.0, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol, 0.5% Triton X-100. Store at -20^oC.

Reaction Buffer: (x5) 250 mM Tris-HCl, pH8.0, 375 mM KCl, 15 mM MgCl₂, 50 mM DTT.

Application: First strand DNA synthesis for cloning and hybridization; RT-PCR.

Quality Control Tests: Activity, SDS-PAGE purity, absence of dsDNases and endonucleases/nickases.

8. Ribonuclease Inhibitor (IRNase) (кат. № IRM-01)

Description: Ribonuclease inhibitor (IRNase) is a protein with molecular weight of 51 kDa. It has an excellent activity to inhibit different RNAses (A, B, C) by binding noncovalently in 1:1 ratio with an association constant 10¹⁴. This product is isolated from human placenta. It is useful in any applications, where the presence of an eucariotic RNAses is a potential problem.

Unit Definition: One unit inhibits 5 ng of RNAse A by 50 % using cytidine 2', 3' - cyclic monophosphat (cCMP) as a substrate.

Concentration: 5 u/ml and 20 u/ml.

Storage (Dilution) Buffer: For 5 u/ml preparation: 25 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM DTT, 0,1 mM EDTA, 50% glycerol, 0.75% Tritone X-100. For 20 u/ml preparation: 25 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM DTT, 1 mM EDTA, 50% glycerol, 0.2% NP-40. Store at -20oC.

Application: RNA purification, first strand DNA synthesis for cloning and hybridization, DNA and RNA sequencing, RT-PCR.

Quality Control Tests: Activity, SDS-PAGE purity, absence of endonucleases/nickases and exonucleases.

9. Thermus aquaticus DNA Polymerase Enhancer (кат. № EPT-01)

Description: Thermus aquaticus DNA Polymerase Enhancer (DPE) is an additive that can greatly increase the yield and specificity of primer extension reactions. DPE destabilizes many mismatched primer-template complexes that would otherwise result in a heterogeneous population of molecules. The addition of DPE generates greater yields in case of limited amounts of amplified product using standard conditions, as well as results in a substantial increase in PCR product specificity.

Concentration: 1 u/ml.

Storage (Dilution) Buffer: 25 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol, 0,75% Triton X-100. Store at -20^oC.

Reaction Conditions: Add 1-2 u of DPE in master mix (without template DNA), incubate 5-10 min at room temperature and then add DNA.

Application: PCR pattern improvement, PCR diagnostic, DNA sequencing improvement.

Quality Control Tests: Activity, SDS-PAGE purity, absence of endonucleases/nickases and exonucleases.

10. Hot-start PCR Compound (HS Compound) (кат. № IPT-01)

Description: “Hot Start” (HS) PCR is commonly used to enhance the specificity and sensitivity of PCR amplification. In many cases, HS PCR has yielded single products in a greater yield than has been possible with conventional PCR. Usually HS PCR is inconvenient, time-consuming and incurs a risk of cross contamination. These inconveniences are overcome by HS Compound. HS Compound is used to block Taq DNA Polymerase activity during set-up of the PCR reactions at ambient temperatures (20-25°C). The inhibition of Taq DNA polymerase is completely reversed when the temperature is raised above 48°C. HS Compound is effective with variety of commercially available Taq DNA Polymerases (native or recombinant). The use of HS Compound significantly improves the specificity of PCR amplification what is especially important for PCR-based diagnostics.

Concentration: 1 u/ml.

Storage Conditions: Store at -20^oC.

Reaction Buffer: The reaction buffer is the same as for the thermostable DNA Polymerase used.

Reaction Conditions: Do not use HS Compound when the PCR annealing cycle temperature is below 48°C. Add 1-2 u of HS Compound in master mix (without template DNA), incubate 5-10 min at room temperature and then add DNA.

Application: PCR, PCR diagnostics.

Quality Control Tests: Activity, SDS-PAGE purity, absence of endonucleases/nickases and exonucleases.

11. T4 DNA Ligase (кат. № LTM-01)

Description: T4 DNA Ligase (recombinant form of the enzyme) catalizes the formation of a phosphodiester bond between the 5'-phosphate and 3'-hydroxyl groups of adjacent nucleotides in DNA or RNA duplex. The enzyme has been shown to join blunt-end and cohesive-end termini as well as repair single stranded nicks in DNA and RNA duplexes, or DNA/RNA hybrids.

Unit Definition: One unit is defined as the amount of the enzyme required to give 50% ligation of HindIII fragments of lambda DNA in 30 minutes at 16^oC in 20 ml of the assay mixture and a 5' DNA termini concentration of 0.12 mM (300 mg/ml). One cohesive end ligation unit equals 0.015 Weiss units.

Concentration: 100 u/ml.

Storage (Dilution) Buffer: 10 mM Tris-HCl, pH 7.5, 50 mM KCl, 0.1 mM EDTA, 1 mM 2-mercaptoethanol, 50% glycerol. Store at -20^oC.

Reaction Buffer (10x): 500 mM Tris-HCl, pH 7.8, 100 mM MgCl₂, 100 mM DTT, 10 mM ATP, 250 mg/ml BSA. Optimal ligation occurs at 16^oC.

Application: DNA ligation.

Quality Control Tests: Activity, SDS-PAGE purity, Blue/White assay, absence of ssDNase, dsDNase, endonucleases/nickases, phosphatases.

12. Uracil-DNA Glycosylase (UDG) (кат. № UEM-01)

Description: E. coli uracil-DNA glycosylase (UDG) - recombinant form of the enzyme, which catalyzes the release of free uracil from uracil-containing DNA. UDG efficiently hydrolyzed uracil from single-stranded or double-stranded DNA, but not from oligomers (6 or fewer bases).

Unit Definition: One unit of activity is the amount of enzyme thet catalyzes the release of 60 pmol of uracil per minute from double-stranded, uracil-containing DNA. Activity is measured by release of [³H]-uracil in a 50 ml reaction containing 0.2 mg DNA (8.7x10⁴ cpm/mg) for 30 minutes at 37^oC.

Concentration: 1 u/ml.

Storage (Dilution) Buffer: 25 mM Tris-HCl, pH8.0, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.5% Triton X-100, 50% glycerol. Store at -20^oC.

Application: PCR, RT-PCR, site-directed mutagenesis, as a probe for protein-DNA interaction studies.

Quality Control Tests: Activity, SDS-PAGE purity, absence of endonucleases/nickases and exonucleases.

13. RT PCR one tube (кат. № RPM-01)

Reagents for RT PCR one tube

1. Reaction buffer х5
2. dNTP 5mM
3. Enzyme mix
4. H₂O (depc)

Reaction mix for RT PCR one tube

Total RNA 1-5 mkl
Reaction buffer х5 5 mkl
dNTP 5mM 2 mkl
Primers:
Rev 20 pmol
For 10 pmol
Enzymes (mix) 1 mkl
H₂O up to 25 mkl

Mix thoroughly! Put the tubes in the thermocycler.

Use the next programme:

I. 42 ⁰С – 30 min
II. 95⁰C – 10 min
III. 25-35 amplification cycles
IV. 72 ⁰С – 5 min

10 ⁰С – storage.

14. Thermus thermophilus DNA Polymerase, modified (Tth-HighRT) (кат. № PTT-31)

Description: Tth DNA Polymerase, modified (Tth-HighRT) - recombinant modified form of the enzyme from the thermophilic eubacterium Thermus thermophilus strain HB8. This enzyme can be used to reverse transcribe RNA efficiently in the presence of Mn²⁺ ions at elevated temperature (70^oC) and subsequently acts as a DNA polymerase to perform PCR process in a single reaction tube.

Unit Definition: One unit of activity is the amount of enzyme required to incorporate 10 nmoles of dNTP into acid-insoluble material in 30 minutes at 72^oC.

Concentration: 5 u/ml.

Storage (Dilution) Buffer: 25 mM Tris-HCl, pH8.0, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol, 0.5% Triton X-100. Store at -20^oC.

Reaction Buffer (10x) (Mn²⁺ minus): 500 mM bicine-KOH (pH 8.3), 1 M KOAc (pH 7.5). Solution of 25 mM Mn(OAc)₂ is included.

Attention! The Reaction Buffer does not contain Mn(OAc)₂. Add demanded quantity of stock solution 25 mM Mn(OAc)₂ to master mix to final concentration 2.5 mM.

Application: PCR, RT-PCR, Sequencing.

Quality Control Tests: Activity, SDS-PAGE purity, absence of endonucleases/nickases and exonucleases.

15. DNA Polymerase I Large (Klenow) Fragment (кат. № PEM-01)

Description: DNA Polymerase I Large (Klenow) Fragment (recombinant) exhibits the 5'-3' polymerase and the 3'-5' exonuclease activities of DNA Polymerase I, E. coli, but lacks its 5'-3' exonuclease activity.

Unit Definition: One unit of activity is the amount of enzyme required to incorporate 10 nmoles of (³H)-dTTP in thirty minutes at 37^oC.

Concentration: 2u./ml.

Storage (Dilution) Buffer: 25 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.75% Triton X-100, 50% glycerol. Store at -20^oC.

Reaction Buffer: (10x) 500 mM Tris-HCl, pH 7.5, 50 mM MgCl₂, 10 mM DTT.

Application: DNA labeling

Quality Control Tests: Activity, SDS-PAGE purity, absence of endonucleases/nickases.

16. Reverse Transcriptase Enhancer (кат. № EPM-01)

Description: Reverse Transcriptase Enhancer (RT Enhancer) is an additive that can greatly increase the yield and specificity of reverse transcription. RT-Enhancer destabilizes hairpin RNA structure and activates annealing of the primer to RNA. The addition of RT-Enhancer generates greater yields in case of limited amounts of RNA using standard conditions, as well as results in a substantial increase in RT specificity and size of resulted product.

Concentration: 1 u/ml, 2 u/ml.

Storage (Dilution) Buffer: 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol, 0,5% Triton X-100. Store at -20^oC.

Reaction Conditions: Use 1 or 2 u of RT-Enhancer in reaction.

Application: First strand DNA synthesis for cloning and hybridization; DNA and RNA sequencing; RT-PCR.

Quality Control Tests: Activity, SDS-PAGE purity, absence of endonucleases/nickases and exonucleases

17. Thermus thermophilus Inorganic Pyrophosphatase (кат. № PPTT-01)

Description: Tth inorganic pyrophosphatase (PPi) - recombinant form of the enzyme from the thermophilic eubacterium Thermus thermophilus strain HB8. This enzyme catalyzes the hydrolysis of inorganic pyrophosphate to form orthophosphate:

Unit Definition: One unit of activity is the amount of enzyme that will generate 40 nmoles of phosphate per minute from pyrophosphate under standard reaction conditions (a 10 minute reaction at 75^oC in 50 mM Tris-HCl, pH 8.8, 5 mM MgCl₂, 1,6 mM PPi, reaction volume of 50 mkl).

Concentration: 1 u/ml.

Storage (Dilution) Buffer: 50 mM Tris-HCl, pH8.0, 100 mM NaCl, 0.01 mM EDTA, 0.1 mM DTT and 50% glycerol. Store at -20^oC.

Reaction Buffer: 50 mM Tris-HCl, pH 8.8, 5 mM MgCl₂ or buffer for sequencing protocol.

Application: PCR, RT-PCR, sequencing.

Quality Control Tests: Activity, SDS-PAGE purity, absence of endonucleases/nickases and exonucleases.

18. T4 Polynucleotide Kinase (кат. № KTM-01)

Description: T4 polynucleotide kinase - recombinant form of the enzyme purified from an E. coli strain carrying overproducing plasmid. This enzyme catalyzes transfer of phosphate group from ATP to 5'-OH termini of DNA, RNA, oligonucleotides or nucleoside 3'-monophosphate as well as the exchange of 5'-terminal phosphate groups. It also has 3'-phosphatase activity.

Unit Definition: One unit of activity is the amount of enzyme required to transfer 1 nmoles of phosphate from [g-³²P]-ATP to 5'-OH DNA in 30 minutes at 37^oC. Activity assay is performed in mix containing 100 mM Tris-HCl (pH 8.0), 10 mM MgCl₂, 5 mM DTT, 0.5 mM 5'-OH DNA and 0.05 mM [³²P]-ATP.

Concentration: 5 u/ml, 20 u/ml.

Storage (Dilution) Buffer: 50 mM Tris-HCl, pH7.6, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT and 50% glycerol. Store at -20^oC.

Reaction Buffer (x10): 400 mM Tris-HCl, pH 7.5, 100 mM MgCl₂, 50 mM DTT.

Application: 5'-termini labeling of nucleic acids, DNA sequencing.

Quality Control Tests: Activity, SDS-PAGE purity, absence of endonucleases/nickases, exonucleases and ribonucleases.

19. Thermus aquaticus Structure-Specific Endonucleolytic Enzyme (CleavTaq) (кат. № PTT-08)

Description: Thermus aquaticus Structure-Specific Endonucleolytic Enzyme (CleavTaq) (5' nuclease of Taq DNA Polymerase, recombinant modified enzyme) is a thermostable nuclease that recognizes and cleaves the junctions between single- and double-stranded DNA regions in partially duplexed DNA molecules. The substrate - DNA structure consisting of partially duplexed double-stranded hairpin regions interspersed with single-stranded regions is formed as a result of denaturation and subsequent cooling of double stranded DNA to an intermediate temperature.

Unit Definition: The activity of enzyme was determined by digestion of an oligonucleotide substrate (5'-TGGTCG

CTGTCTCGCTGAAAGCGAGACAGCGTG-3'), which folds to create a 12-bp stem with a 3-nucleotide loop. Reaction mixtures, containing enzyme and 20 pmol of this substrate in 20 ml of 10 mM MOPS, pH 7.5, 1 mM MnCl₂, were incubated at 50^oC for 2 min. One unit of enzymatic activity is defined as the amount that digests 3.7 pmol of this substrate under conditions of vast substrate excess in 1 min.

Concentration: 5 u/ml.

Storage (Dilution) Buffer: 25 mM Tris-HCl, pH8.0, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol, 0.75% Triton X-100. Store at -20^oC.

Reaction Buffer: (x10) 100 mM Tris-HCl, 20 mM MnCl₂, 1% Triton X-100 for 0.1 - 1.0 kbp DNA fragments, or 100 mM Tris-HCl, pH 7.5, 20 mM MnCl₂, 100 mM MgCl₂, 1% Triton X-100 for 0.5-2.0 kbp DNA fragments. Reaction can be performed also in the buffers used for Taq DNA Polymerase.

Application: CFLP (Cleavage Fragment Lenght Polymorfism) technology; gene polymorphism detection.

Quality Control Tests: Activity, SDS-PAGE purity.

20. Long PCR. (кат. № PTT-10)

Объем реакционной смеси 50 мкл:

1. Taq pol PCR buffer x10, содержащий 25 мМ MgCl₂
2. MgCl₂ до 5 мМ
3. Трифосфаты до 400 мкМ
4. Праймеры по 20 пмоль
5. Количество матрицы необходимо подбирать в каждом конкретном случае. Имеет смысл начать с двух диапазонов: 1-10 нг, 50-200 нг. Матрицы для LPCR необходимо брать примерно в 2-4 раза больше, чем для PCR коротких фрагментов.
6. Taqm₂-pol 10 единиц
7. Taqm₂⁺-pol 0,6 единиц.

Реакционная смесь готовится в охлажденных во льду пробирках. Все добавляемые ингредиенты также необходимо охладить. Готовятся две смеси:

Смесь 1.

Трифосфаты
ДНК
Праймеры
Вода до 25 мкл

Смесь 2.

Taq pol PCR buffer x10
MgCl₂
Taqm₂-pol 10 единиц
Taqm₂⁺-pol 0.6 единиц
Вода до 25 мкл

Смеси также охлаждают во льду и смешивают непосредственно перед помещением в амплификатор. Таким способом достигается предотвращение работы полимераз до начала реакции амплификации. Это особенно важно в отношении Taqm₂⁺-pol

Режим PCR:

Денатурация 95⁰С 30 сек.

Отжиг праймеров 1 мин.

Элонгация цепи 72⁰С. Время элонгации зависит от длины амплифицируемого фрагмента и приблизительно рассчитывается исходя из значений 1 мин. на 1.3-1.5 кb амплифицируемого фрагмента. Время, рассчитанное таким образом используется в первых десяти циклах. Для каждых последующих пяти циклов добавляется еще по 20 сек.

Общее количество циклов 30-35.