To detect the frequency of vanA, vanB, vanC, vanD genes, multiplex-PCR using specific primers was used (Table 1 ). Extracted DNA was done as described above. The PCR mixture included 3.5 µl of double distilled water, 12.5 µl of Taq 2X master mix (Ampliqon, Cat No. A190303, Denmark), 0.5 µl of each primer (10 pmol/ml, Table 1 ), and 5 µl of DNA. Cycling conditions were done as follows: 1 cycle of initial denaturation at 94 oC for 3 min, 35 cycles of initial denaturation at 94 oC for 1 min, annealing at 54 oC for 1 min, extension at 72 oC for 1 min, and 1 cycle of final extension at 72 °C for 7 min.
Taq 2x master mix
Manufactured by Ampliqon
Sourced in Germany
Taq 2x master mix is a pre-formulated solution containing Taq DNA polymerase, necessary reaction buffers, and dNTPs. It is designed to simplify and streamline PCR setup.
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Lab products found in correlation
6 protocols using taq 2x master mix
1
Multiplex-PCR Detection of Vancomycin Resistance Genes
2
Genotyping of ACSF3 and CASC16 SNPs
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To determine the genotype frequency of rs4782447-ACSF3 and rs4784227-CASC16 SNPs, ARMS-PCR was used. PCR amplifications for rs4782447-ACSF3 and rs4784227-CASC16 have been carried out in a 10 μl final volume per reaction containing three µl Taq 2x master mix (Ampliqon, Germany), one µl of each primer (10 µM) and 100 ng DNA. The primers used for detection of rs4782447-ACSF3 and rs4784227-CASC16 SNPs are listed in Table 1(Tab. 1) . The ARMS-PCR condition for rs4782477 was as follows: initial denaturation at 94 °C for five minutes, after that 35 cycles including denaturation at 94 °C for 25 seconds, annealing at 59 °C for 25 seconds, an extension at 72 °C for 30 seconds followed by 72 °C for seven minutes as the final extension step. Moreover, ARMS-PCR condition for rs4784227-CASC16 was the same as rs4782477 with a different annealing temperature of 71 °C. The DNA fragments of PCR products were detected using electrophoresis in 2 % agarose gel.
3
Molecular Detection of E. granulosus NAD1
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In the polymerase chain reaction (PCR) method, primers NAD1F 5′ - TATTCTCARTYTCGYAAGGGHCC - 3′ and NAD1R 5′ -AACCATTTCTTGAAGTTAACAGCAGCATC - 3′ amplifying the E. granulosus NADH dehydrogenase subunit 1 (NAD1) gene region were used (6 (link)). The reaction was adjusted to a total volume of 50 μL, containing 25 μL of Taq 2x Master Mix (with 12.5 mM MgCl2) (Ampliqon), 0.5 mM of MgCl2, 0.2 μM from each primer (Sentegen), and 4 μL of sample DNA.
Reactions were run on an Applied Biosystems SimpliAmp Thermal Cycler PCR instrument. PCR was programmed for 45 cycles of 40 s at 95 °C, 40 s at 50°C, and 65 s at 72 °C. In addition to the PCR process, a 4-min denaturation step at 95 °C was performed before the first cycle, as well as an extension phase at 72 °C for 10 min following the last cycle. In order to display the results, 15 μL of the obtained reaction products were run in gel electrophoresis and visualized in the UVP Gel documentation system.
Reactions were run on an Applied Biosystems SimpliAmp Thermal Cycler PCR instrument. PCR was programmed for 45 cycles of 40 s at 95 °C, 40 s at 50°C, and 65 s at 72 °C. In addition to the PCR process, a 4-min denaturation step at 95 °C was performed before the first cycle, as well as an extension phase at 72 °C for 10 min following the last cycle. In order to display the results, 15 μL of the obtained reaction products were run in gel electrophoresis and visualized in the UVP Gel documentation system.
4
ARMS-PCR Genotyping of PICK1 and BDNF SNPs
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To determine the genotype frequency of PICK1-rs713729, PICK1-rs2076369 and BDNF-rs6265 an ARMS-PCR method was used. Speci c primers for PCR ampli cation were designed via web tools, such as Primer1 and also WASP (web-based allele-speci c primer designing tool) [28] .
PCR ampli cations for PICK1-rs713729, PICK1-rs2076369 and BDNF-rs6265 were conducted in a 10-15 microliter (μl) volume per reaction, containing 3 µl Taq 2x master mix (Ampliqon, Germany), 10 µM of each primer and 100 nanogram (ng) DNA. Moreover, the speci c primers used to detect PICK1-rs713729, PICK1-rs2076369 and BDNF-rs6265 SNPs are listed in Table 4. For PICK1-rs2076369, we also used betaine (Ampliqon, Germany) as an enhancer in PCR.
The ARMS-PCRs condition for each primer is as follows, Table 5. In general, initial denaturation at temperature 94 °C for ve minutes, then 35 cycles including denaturation at 94 °C for 25 seconds, annealing at alternative °C for 25 seconds (based on each primer), an elongation at 72 °C for 30 seconds followed by 72 °C for seven minutes as the nal elongation step (Table 5).
Absence or presence of mutant or normal PCR products were detected via gel electrophoresis in 3% agarose gel by ultraviolet (UV) trans illuminator (Gel Doc; U:Genius). Table 4 Primer sequences used for genotyping in ARMS-PCR.
Table 5 The ARMS-PCRs condition for targeted SNPs.
PCR ampli cations for PICK1-rs713729, PICK1-rs2076369 and BDNF-rs6265 were conducted in a 10-15 microliter (μl) volume per reaction, containing 3 µl Taq 2x master mix (Ampliqon, Germany), 10 µM of each primer and 100 nanogram (ng) DNA. Moreover, the speci c primers used to detect PICK1-rs713729, PICK1-rs2076369 and BDNF-rs6265 SNPs are listed in Table 4. For PICK1-rs2076369, we also used betaine (Ampliqon, Germany) as an enhancer in PCR.
The ARMS-PCRs condition for each primer is as follows, Table 5. In general, initial denaturation at temperature 94 °C for ve minutes, then 35 cycles including denaturation at 94 °C for 25 seconds, annealing at alternative °C for 25 seconds (based on each primer), an elongation at 72 °C for 30 seconds followed by 72 °C for seven minutes as the nal elongation step (Table 5).
Absence or presence of mutant or normal PCR products were detected via gel electrophoresis in 3% agarose gel by ultraviolet (UV) trans illuminator (Gel Doc; U:Genius). Table 4 Primer sequences used for genotyping in ARMS-PCR.
Table 5 The ARMS-PCRs condition for targeted SNPs.
5
Genotyping of PICK1 and BDNF Variants
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To determine the genotype frequency of PICK1-rs713729, PICK1-rs2076369 and BDNF-rs6265 an ARMS-PCR method was used. Speci c primers for PCR ampli cation were designed via web tools, such as Primer1 and also WASP (web-based allele-speci c primer designing tool) [28] .
PCR ampli cations for PICK1-rs713729, PICK1-rs2076369 and BDNF-rs6265 were conducted in a 10-15 microliter (μl) volume per reaction, containing 3 µl Taq 2x master mix (Ampliqon, Germany), 10 µM of each primer and 100 nanogram (ng) DNA. Moreover, the speci c primers used to detect PICK1-rs713729, PICK1-rs2076369 and BDNF-rs6265 SNPs are listed in Table 4. For PICK1-rs2076369, we also used betaine (Ampliqon, Germany) as an enhancer in PCR.
The ARMS-PCRs condition for each primer is as follows, Table 5. In general, initial denaturation at temperature 94 °C for ve minutes, then 35 cycles including denaturation at 94 °C for 25 seconds, annealing at alternative °C for 25 seconds (based on each primer), an elongation at 72 °C for 30 seconds followed by 72 °C for seven minutes as the nal elongation step (Table 5).
PCR ampli cations for PICK1-rs713729, PICK1-rs2076369 and BDNF-rs6265 were conducted in a 10-15 microliter (μl) volume per reaction, containing 3 µl Taq 2x master mix (Ampliqon, Germany), 10 µM of each primer and 100 nanogram (ng) DNA. Moreover, the speci c primers used to detect PICK1-rs713729, PICK1-rs2076369 and BDNF-rs6265 SNPs are listed in Table 4. For PICK1-rs2076369, we also used betaine (Ampliqon, Germany) as an enhancer in PCR.
The ARMS-PCRs condition for each primer is as follows, Table 5. In general, initial denaturation at temperature 94 °C for ve minutes, then 35 cycles including denaturation at 94 °C for 25 seconds, annealing at alternative °C for 25 seconds (based on each primer), an elongation at 72 °C for 30 seconds followed by 72 °C for seven minutes as the nal elongation step (Table 5).
6
Genotyping SNPs via ARMS-PCR
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To determine the genotype frequency of PICK1-rs713729, PICK1-rs2076369 and BDNF-rs6265 an ARMS-PCR method was used. Speci c primers for PCR ampli cation were designed via web tools, such as Primer1 and also WASP (web-based allele-speci c primer designing tool) [28] .
PCR ampli cations for PICK1-rs713729, PICK1-rs2076369 and BDNF-rs6265 were conducted in a 10-15 microliter (μl) volume per reaction, containing 3 µl Taq 2x master mix (Ampliqon, Germany), 10 µM of each primer and 100 nanogram (ng) DNA. Moreover, the speci c primers used to detect PICK1-rs713729, PICK1-rs2076369 and BDNF-rs6265 SNPs are listed in Table4. For PICK1-rs2076369, we also used betaine (Ampliqon, Germany) as an enhancer in PCR.
The ARMS-PCRs condition for each primer is as follows, Table 5. In general, initial denaturation at temperature 94 °C for ve minutes, then 35 cycles including denaturation at 94 °C for 25 seconds, annealing at alternative °C for 25 seconds (based on each primer), an elongation at 72 °C for 30 seconds followed by 72 °C for seven minutes as the nal elongation step (Table 5).
Absence or presence of mutant or normal PCR products were detected via gel electrophoresis in 3% agarose gel by ultraviolet (UV) trans illuminator (Gel Doc; U:Genius).
PCR ampli cations for PICK1-rs713729, PICK1-rs2076369 and BDNF-rs6265 were conducted in a 10-15 microliter (μl) volume per reaction, containing 3 µl Taq 2x master mix (Ampliqon, Germany), 10 µM of each primer and 100 nanogram (ng) DNA. Moreover, the speci c primers used to detect PICK1-rs713729, PICK1-rs2076369 and BDNF-rs6265 SNPs are listed in Table4. For PICK1-rs2076369, we also used betaine (Ampliqon, Germany) as an enhancer in PCR.
The ARMS-PCRs condition for each primer is as follows, Table 5. In general, initial denaturation at temperature 94 °C for ve minutes, then 35 cycles including denaturation at 94 °C for 25 seconds, annealing at alternative °C for 25 seconds (based on each primer), an elongation at 72 °C for 30 seconds followed by 72 °C for seven minutes as the nal elongation step (Table 5).
Absence or presence of mutant or normal PCR products were detected via gel electrophoresis in 3% agarose gel by ultraviolet (UV) trans illuminator (Gel Doc; U:Genius).
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