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  • 99
    Thermo Fisher geneamp pcr system 9700 thermocycler
    Geneamp Pcr System 9700 Thermocycler, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1993 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/geneamp pcr system 9700 thermocycler/product/Thermo Fisher
    Average 99 stars, based on 1993 article reviews
    Price from $9.99 to $1999.99
    geneamp pcr system 9700 thermocycler - by Bioz Stars, 2020-09
    99/100 stars
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    94
    Bio-Rad thermocycler
    Thermocycler, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 6886 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/thermocycler/product/Bio-Rad
    Average 94 stars, based on 6886 article reviews
    Price from $9.99 to $1999.99
    thermocycler - by Bioz Stars, 2020-09
    94/100 stars
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    94
    Thermo Fisher thermocycler
    Nested/PCR-RFLP of ( A ) Plasmodium falciparum samples, two patients from Pará State (Pf – PA) and Acre State (Pf – AC) and Pf 3D7 culture (diluted 1:100); ( B ) Plasmodium brasilianum/Plasmodium malariae , two NHPs (Pbr 2434 and Pbr 2620), two patients (Pm P3 and Pm l11) and Plasmodium brasilianum from MR4 (diluted 1:100 in water); ( C ) Negative controls of PCR: two uninfected humans (UH - 1 and 2), one uninfected NHP (UNHP), and negative control of PCR (NC - without DNA). Reactions were performed simultaneously in the same <t>thermocycler</t> and splited in different agarose gels. 3% Agarose gelstained with ethidium bromide. MM: 1 kb Plus Ladder. D: Digested; ND: Non Digested.
    Thermocycler, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 6319 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/thermocycler/product/Thermo Fisher
    Average 94 stars, based on 6319 article reviews
    Price from $9.99 to $1999.99
    thermocycler - by Bioz Stars, 2020-09
    94/100 stars
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    94
    Eppendorf AG thermocycler
    Sensitivity assessment of the LAMP assay for Loa loa using serial dilutions of genomic DNA by the addition of SYBR Green I or by visualization on agarose gel stained with ethidium bromide. (A) Sensitivity assessment performed with a <t>thermocycler.</t> (B) Sensitivity assessment performed with a heating block. Lane Loa: genomic DNA from Loa loa (5 ng); lanes 10 −1 –10 −13 : 10-fold serially dilutions; lanes N, N1, N2, N3: negative controls (no DNA template). Lane M: 50 bp DNA ladder (Molecular weight marker XIII, Roche).
    Thermocycler, supplied by Eppendorf AG, used in various techniques. Bioz Stars score: 94/100, based on 4704 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/thermocycler/product/Eppendorf AG
    Average 94 stars, based on 4704 article reviews
    Price from $9.99 to $1999.99
    thermocycler - by Bioz Stars, 2020-09
    94/100 stars
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    93
    MJ Research thermocycler
    Optimization of temperature and paper type. (a) Temperature optimization among 65 °C, 68 °C and 70 °C using a conventional <t>thermocycler,</t> as confirmed with 3% w/v agarose gel electrophoresis. (b) Paper type optimization among NC (nitrocellulose), G4 (cellulose grade 4) and G1 (cellulose grade 1) papers, as confirmed with 3% w/v agarose gel electrophoresis.
    Thermocycler, supplied by MJ Research, used in various techniques. Bioz Stars score: 93/100, based on 2250 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/thermocycler/product/MJ Research
    Average 93 stars, based on 2250 article reviews
    Price from $9.99 to $1999.99
    thermocycler - by Bioz Stars, 2020-09
    93/100 stars
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    94
    Bio-Rad cfx96 thermocycler
    Optimization of temperature and paper type. (a) Temperature optimization among 65 °C, 68 °C and 70 °C using a conventional <t>thermocycler,</t> as confirmed with 3% w/v agarose gel electrophoresis. (b) Paper type optimization among NC (nitrocellulose), G4 (cellulose grade 4) and G1 (cellulose grade 1) papers, as confirmed with 3% w/v agarose gel electrophoresis.
    Cfx96 Thermocycler, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 1831 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cfx96 thermocycler/product/Bio-Rad
    Average 94 stars, based on 1831 article reviews
    Price from $9.99 to $1999.99
    cfx96 thermocycler - by Bioz Stars, 2020-09
    94/100 stars
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    92
    Biometra thermocycler
    Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The <t>thermocycler</t> (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.
    Thermocycler, supplied by Biometra, used in various techniques. Bioz Stars score: 92/100, based on 2435 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/thermocycler/product/Biometra
    Average 92 stars, based on 2435 article reviews
    Price from $9.99 to $1999.99
    thermocycler - by Bioz Stars, 2020-09
    92/100 stars
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    93
    Eppendorf AG mastercycler gradient thermocycler
    Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The <t>thermocycler</t> (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.
    Mastercycler Gradient Thermocycler, supplied by Eppendorf AG, used in various techniques. Bioz Stars score: 93/100, based on 1812 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mastercycler gradient thermocycler/product/Eppendorf AG
    Average 93 stars, based on 1812 article reviews
    Price from $9.99 to $1999.99
    mastercycler gradient thermocycler - by Bioz Stars, 2020-09
    93/100 stars
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    94
    MJ Research ptc 200 thermocycler
    Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The <t>thermocycler</t> (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.
    Ptc 200 Thermocycler, supplied by MJ Research, used in various techniques. Bioz Stars score: 94/100, based on 1689 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ptc 200 thermocycler/product/MJ Research
    Average 94 stars, based on 1689 article reviews
    Price from $9.99 to $1999.99
    ptc 200 thermocycler - by Bioz Stars, 2020-09
    94/100 stars
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    92
    MJ Research ptc 100 thermocycler
    Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The <t>thermocycler</t> (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.
    Ptc 100 Thermocycler, supplied by MJ Research, used in various techniques. Bioz Stars score: 92/100, based on 1468 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ptc 100 thermocycler/product/MJ Research
    Average 92 stars, based on 1468 article reviews
    Price from $9.99 to $1999.99
    ptc 100 thermocycler - by Bioz Stars, 2020-09
    92/100 stars
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    92
    Thermo Fisher steponeplus thermocycler
    Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The <t>thermocycler</t> (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.
    Steponeplus Thermocycler, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1096 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/steponeplus thermocycler/product/Thermo Fisher
    Average 92 stars, based on 1096 article reviews
    Price from $9.99 to $1999.99
    steponeplus thermocycler - by Bioz Stars, 2020-09
    92/100 stars
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    90
    Bio-Rad iq5 thermocycler
    Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The <t>thermocycler</t> (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.
    Iq5 Thermocycler, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 90/100, based on 700 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/iq5 thermocycler/product/Bio-Rad
    Average 90 stars, based on 700 article reviews
    Price from $9.99 to $1999.99
    iq5 thermocycler - by Bioz Stars, 2020-09
    90/100 stars
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    91
    Biometra t3 thermocycler
    Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The <t>thermocycler</t> (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.
    T3 Thermocycler, supplied by Biometra, used in various techniques. Bioz Stars score: 91/100, based on 980 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t3 thermocycler/product/Biometra
    Average 91 stars, based on 980 article reviews
    Price from $9.99 to $1999.99
    t3 thermocycler - by Bioz Stars, 2020-09
    91/100 stars
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    91
    Biometra t gradient thermocycler
    Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The <t>thermocycler</t> (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.
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    Nested/PCR-RFLP of ( A ) Plasmodium falciparum samples, two patients from Pará State (Pf – PA) and Acre State (Pf – AC) and Pf 3D7 culture (diluted 1:100); ( B ) Plasmodium brasilianum/Plasmodium malariae , two NHPs (Pbr 2434 and Pbr 2620), two patients (Pm P3 and Pm l11) and Plasmodium brasilianum from MR4 (diluted 1:100 in water); ( C ) Negative controls of PCR: two uninfected humans (UH - 1 and 2), one uninfected NHP (UNHP), and negative control of PCR (NC - without DNA). Reactions were performed simultaneously in the same thermocycler and splited in different agarose gels. 3% Agarose gelstained with ethidium bromide. MM: 1 kb Plus Ladder. D: Digested; ND: Non Digested.

    Journal: Scientific Reports

    Article Title: An assay for the identification of Plasmodium simium infection for diagnosis of zoonotic malaria in the Brazilian Atlantic Forest

    doi: 10.1038/s41598-017-18216-x

    Figure Lengend Snippet: Nested/PCR-RFLP of ( A ) Plasmodium falciparum samples, two patients from Pará State (Pf – PA) and Acre State (Pf – AC) and Pf 3D7 culture (diluted 1:100); ( B ) Plasmodium brasilianum/Plasmodium malariae , two NHPs (Pbr 2434 and Pbr 2620), two patients (Pm P3 and Pm l11) and Plasmodium brasilianum from MR4 (diluted 1:100 in water); ( C ) Negative controls of PCR: two uninfected humans (UH - 1 and 2), one uninfected NHP (UNHP), and negative control of PCR (NC - without DNA). Reactions were performed simultaneously in the same thermocycler and splited in different agarose gels. 3% Agarose gelstained with ethidium bromide. MM: 1 kb Plus Ladder. D: Digested; ND: Non Digested.

    Article Snippet: The PCR assays were performed in a thermocycler (Veriti 96 wells, Applied Biosystems) with the following cycling parameters: an initial denaturation at 94 °C for 2 min followed by 40 cycles of denaturation at 94 °C for 30 sec, annealing at 54 °C for 20 sec and extension at 72 °C for 30 sec followed by a final extension incubation at 72 °C for 2 min.

    Techniques: Nested PCR, Polymerase Chain Reaction, Negative Control

    Differential diagnosis of Plasmodium simium infection by nested/PCR followed by a digestion with Hpy CH4III restriction enzyme of 9 infected human samples from Atlantic Forest in Rio de Janeiro/RJ (H2 – H9) and one from Amazon endemic region (H1) according to Additional file 1. 3% agarose gel stained with ethidium bromide. MM:1 kb Plus Ladder (ThermoFischer, Calrsbad, CA, USA). Reactions were performed simultaneously in the same thermocycler and splited in different gels.D: Digested; ND: Non Digested. PC Pv: Positive Control for P. vivax , PC Ps: positive control for P. simium . NC: Negative Control (without DNA).

    Journal: Scientific Reports

    Article Title: An assay for the identification of Plasmodium simium infection for diagnosis of zoonotic malaria in the Brazilian Atlantic Forest

    doi: 10.1038/s41598-017-18216-x

    Figure Lengend Snippet: Differential diagnosis of Plasmodium simium infection by nested/PCR followed by a digestion with Hpy CH4III restriction enzyme of 9 infected human samples from Atlantic Forest in Rio de Janeiro/RJ (H2 – H9) and one from Amazon endemic region (H1) according to Additional file 1. 3% agarose gel stained with ethidium bromide. MM:1 kb Plus Ladder (ThermoFischer, Calrsbad, CA, USA). Reactions were performed simultaneously in the same thermocycler and splited in different gels.D: Digested; ND: Non Digested. PC Pv: Positive Control for P. vivax , PC Ps: positive control for P. simium . NC: Negative Control (without DNA).

    Article Snippet: The PCR assays were performed in a thermocycler (Veriti 96 wells, Applied Biosystems) with the following cycling parameters: an initial denaturation at 94 °C for 2 min followed by 40 cycles of denaturation at 94 °C for 30 sec, annealing at 54 °C for 20 sec and extension at 72 °C for 30 sec followed by a final extension incubation at 72 °C for 2 min.

    Techniques: Infection, Nested PCR, Agarose Gel Electrophoresis, Staining, Positive Control, Negative Control

    Nested/PCR-RFLP of P. vivax DNA samples. 15 DNA samples from P. vivax infected individuals from different parts of Amazon: Porto Velho/Rondônia State, Brazil (PvPV/RO1 and 2), Guyana (PvGuy), Ariquemes/Rondônia State, Brazil (PvAri/RO), Venezuela (PvVen), French Guiana (PvFrGui), Novo progresso/Pará State, Brazil (PvNP/PA), Rio Pardo/Amazonas State, Brazil (PvRP/AM1, 2 and 3), Humaita/Amazonas State, Brazil (PvHu/Am1, 2, 3 and 4) and unknown city in Amazonas State, Brazil (PvAM) were used for Nested PCR amplification followed by digestion with Hpy CH4III restriction enzyme. 3% Agarose gel stained with ethidium bromide. Name tags above gels indicated the patients according to Additional file 2. Reactions were performed simultaneously in the same thermocycler and splited in different gels. MM: 1 kb Plus Ladder. D: Digested (8 μL of digestion); ND: Non Digested (the equivalent amount of PCR product used in the digestion, 6.5 μL of samples and 5 μL of controls); PC Pv: positive control of P. vivax (pool of samples from infected patient from Amazonia); PC Ps: positive control of P. simium ( Alouatta g. clamitans infected with P. simium ); NC: negative Control.

    Journal: Scientific Reports

    Article Title: An assay for the identification of Plasmodium simium infection for diagnosis of zoonotic malaria in the Brazilian Atlantic Forest

    doi: 10.1038/s41598-017-18216-x

    Figure Lengend Snippet: Nested/PCR-RFLP of P. vivax DNA samples. 15 DNA samples from P. vivax infected individuals from different parts of Amazon: Porto Velho/Rondônia State, Brazil (PvPV/RO1 and 2), Guyana (PvGuy), Ariquemes/Rondônia State, Brazil (PvAri/RO), Venezuela (PvVen), French Guiana (PvFrGui), Novo progresso/Pará State, Brazil (PvNP/PA), Rio Pardo/Amazonas State, Brazil (PvRP/AM1, 2 and 3), Humaita/Amazonas State, Brazil (PvHu/Am1, 2, 3 and 4) and unknown city in Amazonas State, Brazil (PvAM) were used for Nested PCR amplification followed by digestion with Hpy CH4III restriction enzyme. 3% Agarose gel stained with ethidium bromide. Name tags above gels indicated the patients according to Additional file 2. Reactions were performed simultaneously in the same thermocycler and splited in different gels. MM: 1 kb Plus Ladder. D: Digested (8 μL of digestion); ND: Non Digested (the equivalent amount of PCR product used in the digestion, 6.5 μL of samples and 5 μL of controls); PC Pv: positive control of P. vivax (pool of samples from infected patient from Amazonia); PC Ps: positive control of P. simium ( Alouatta g. clamitans infected with P. simium ); NC: negative Control.

    Article Snippet: The PCR assays were performed in a thermocycler (Veriti 96 wells, Applied Biosystems) with the following cycling parameters: an initial denaturation at 94 °C for 2 min followed by 40 cycles of denaturation at 94 °C for 30 sec, annealing at 54 °C for 20 sec and extension at 72 °C for 30 sec followed by a final extension incubation at 72 °C for 2 min.

    Techniques: Nested PCR, Infection, Amplification, Agarose Gel Electrophoresis, Staining, Polymerase Chain Reaction, Positive Control, Negative Control

    Differential Diagnosis of Plasmodium simium infection by nested/PCR followed by a digestion with Hpy CH4III restriction enzyme of 16 non-human primate samples: ( A ) 2 captive NHP from Rio de Janeiro/RJ ( Sapajus xanthosternos 2098; Cacajao melanocephalus 2302), one free-living Alouatta g. clamitans from Rio de Janeiro State (3636) and 6 free-living Alouatta g. clamitans from Joinville/SC, Brazil (J9, J11, J15, J20, J22, J25); ( B ) 7 Alouatta g. clamitans from CEPESBI, Indaial, SC, Brazil (Bl3, Bl6, Bl10, Bl28, Bl61, Bl64, Bl69), *Captive NHPs, all the other were free-living. More details about each sample see Additional file 1. 3% agarose gel stained with ethidium bromide. MM:1 kb Plus Ladder (ThermoFischer). Reactions were performed simultaneously in the same thermocycler and splited in different gels. PC Pv: Positive Control for P. vivax , PC Ps: positive control for P. simium . NC: Negative Control (without DNA).

    Journal: Scientific Reports

    Article Title: An assay for the identification of Plasmodium simium infection for diagnosis of zoonotic malaria in the Brazilian Atlantic Forest

    doi: 10.1038/s41598-017-18216-x

    Figure Lengend Snippet: Differential Diagnosis of Plasmodium simium infection by nested/PCR followed by a digestion with Hpy CH4III restriction enzyme of 16 non-human primate samples: ( A ) 2 captive NHP from Rio de Janeiro/RJ ( Sapajus xanthosternos 2098; Cacajao melanocephalus 2302), one free-living Alouatta g. clamitans from Rio de Janeiro State (3636) and 6 free-living Alouatta g. clamitans from Joinville/SC, Brazil (J9, J11, J15, J20, J22, J25); ( B ) 7 Alouatta g. clamitans from CEPESBI, Indaial, SC, Brazil (Bl3, Bl6, Bl10, Bl28, Bl61, Bl64, Bl69), *Captive NHPs, all the other were free-living. More details about each sample see Additional file 1. 3% agarose gel stained with ethidium bromide. MM:1 kb Plus Ladder (ThermoFischer). Reactions were performed simultaneously in the same thermocycler and splited in different gels. PC Pv: Positive Control for P. vivax , PC Ps: positive control for P. simium . NC: Negative Control (without DNA).

    Article Snippet: The PCR assays were performed in a thermocycler (Veriti 96 wells, Applied Biosystems) with the following cycling parameters: an initial denaturation at 94 °C for 2 min followed by 40 cycles of denaturation at 94 °C for 30 sec, annealing at 54 °C for 20 sec and extension at 72 °C for 30 sec followed by a final extension incubation at 72 °C for 2 min.

    Techniques: Infection, Nested PCR, Agarose Gel Electrophoresis, Staining, Positive Control, Negative Control

    Sensitivity assessment of the LAMP assay for Loa loa using serial dilutions of genomic DNA by the addition of SYBR Green I or by visualization on agarose gel stained with ethidium bromide. (A) Sensitivity assessment performed with a thermocycler. (B) Sensitivity assessment performed with a heating block. Lane Loa: genomic DNA from Loa loa (5 ng); lanes 10 −1 –10 −13 : 10-fold serially dilutions; lanes N, N1, N2, N3: negative controls (no DNA template). Lane M: 50 bp DNA ladder (Molecular weight marker XIII, Roche).

    Journal: PLoS ONE

    Article Title: Development of a Highly Sensitive Loop-Mediated Isothermal Amplification (LAMP) Method for the Detection of Loa loa

    doi: 10.1371/journal.pone.0094664

    Figure Lengend Snippet: Sensitivity assessment of the LAMP assay for Loa loa using serial dilutions of genomic DNA by the addition of SYBR Green I or by visualization on agarose gel stained with ethidium bromide. (A) Sensitivity assessment performed with a thermocycler. (B) Sensitivity assessment performed with a heating block. Lane Loa: genomic DNA from Loa loa (5 ng); lanes 10 −1 –10 −13 : 10-fold serially dilutions; lanes N, N1, N2, N3: negative controls (no DNA template). Lane M: 50 bp DNA ladder (Molecular weight marker XIII, Roche).

    Article Snippet: To establish the standard protocol for the LAMP method, the reaction mixture was incubated in a conventional heating block (K Dry-Bath) or in a thermocycler (Mastercycler Gradient-96well; Eppendorf) at a range of temperatures (61, 63 and 65°C) for 60 min to optimize the reaction conditions and then heated at 80°C for 5 min to terminate the reaction.

    Techniques: Lamp Assay, SYBR Green Assay, Agarose Gel Electrophoresis, Staining, Blocking Assay, Molecular Weight, Marker

    Optimization of temperature and paper type. (a) Temperature optimization among 65 °C, 68 °C and 70 °C using a conventional thermocycler, as confirmed with 3% w/v agarose gel electrophoresis. (b) Paper type optimization among NC (nitrocellulose), G4 (cellulose grade 4) and G1 (cellulose grade 1) papers, as confirmed with 3% w/v agarose gel electrophoresis.

    Journal: Scientific Reports

    Article Title: Simpler, Faster, and Sensitive Zika Virus Assay Using Smartphone Detection of Loop-mediated Isothermal Amplification on Paper Microfluidic Chips

    doi: 10.1038/s41598-018-30797-9

    Figure Lengend Snippet: Optimization of temperature and paper type. (a) Temperature optimization among 65 °C, 68 °C and 70 °C using a conventional thermocycler, as confirmed with 3% w/v agarose gel electrophoresis. (b) Paper type optimization among NC (nitrocellulose), G4 (cellulose grade 4) and G1 (cellulose grade 1) papers, as confirmed with 3% w/v agarose gel electrophoresis.

    Article Snippet: Temperature optimization The optimum temperature was evaluated by conventionally amplifying the NTC and target at three different temperatures, 65 °C, 68 °C, and 70 °C for 30 minutes using a thermocycler (MJ Research, Waltham, MA, USA), followed by refrigeration at 4 °C in preparation for gel electrophoresis confirmation.

    Techniques: Agarose Gel Electrophoresis

    Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.

    Journal: BMC Microbiology

    Article Title: Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism

    doi: 10.1186/1471-2180-4-21

    Figure Lengend Snippet: Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.

    Article Snippet: The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C.

    Techniques: Plasmid Preparation, Polymerase Chain Reaction, Amplification, Size-exclusion Chromatography, Agarose Gel Electrophoresis, Purification, Incubation, Ligation, Clone Assay