Conventional PCR was used to identify genes linked to the apoptosis process (avrA), potential survival under oxidative stress (sodC), invasion (invA), adhesion, and biofilm formation (agfA, sefA, and lpfA) and quorum sensing (luxS) (Table 2 ).The PCR reaction preparation consisted of 12.5 µL of GoTaq® Green Master Mix (Promega®), 1 µL of DNA at 10 ng/µL, 1 µL of the gene-specific primer pairs (Table 2 ), and 10.5 µL of Milli-Q® Water (Merck®, Darmstadt, Germany). The microtubes were transferred to a thermal cycler (Eppendorf®, Hamburg, Germany) for amplification: an initial denaturation cycle at 94 °C for 5min, 35cycles of denaturation at 94 °C for 45 s, annealing for 30 s at 58 °C (invA); 50 °C (sefA and 1pfA), 66 °C (agfA) or 62 °C (avrA, sodC and luxS); extension at 72 °C for 90 s, and a final extension at 72 °C for 10 min. The positive reaction control used was the strain S. Enteritidis ATCC13076 and, as negative control, sterile ultrapure water. Agarose gels (Afllymetrix®, Santa Clara, CA, USA) were stained with SYBR® Safe DNA gel stain solution (Invitrogen®) and visualized under UV light on a transilluminator (Loccus Biotechnology®, São Paulo, Brazil) after 90 min of running the gel at 100 W, 80 V, and 80 A.
Sybr safe dna gel stain solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
SYBR® Safe DNA gel stain solution is a fluorescent dye used for the detection of DNA in agarose or polyacrylamide gels. It is a sensitive and safe alternative to traditional DNA stains like ethidium bromide.
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2 protocols using sybr safe dna gel stain solution
1
Identifying Virulence Genes in Bacteria
2
Multiplex PCR for C. jejuni Identification
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The identification of C. jejuni strains was performed by multiplex PCR (HARMON; RAMSOM; WESLEY, 1997) DNA was extracted with the aid of commercially available kit (Wizard® Genomic DNA Purification Kit -Promega) following the protocol supplied by the manufacturer.
The preparation of PCR consisted of 20 picomoles of primer C1 and C4, 40 picomoles of primers pg3 and pg50 (Table 1), 10 mM of Tris-HCl, 50 mM of KCl, 200 μM of each deoxynucleotide triphosphates (DNTPs), 5.5 mM of MgCl2 and 1,25 U of Taq DNA polymerase and 20 ng of DNA (Invitrogen®). The amplification was according to the following steps: 1 initial denaturation cycle at 94 °C for 4 minutes; 25 cycles of: denaturation at 94 °C for 1 minute, annealing at 47 °C for 1 minute and extension at 72 ºC for 1 minute, followed by a final extension cycle at 72 ºC for 7 minutes (HARMON; RAMSOM; WESLEY, 1997) (link).
The amplified products (8μL) were submitted to electrophoresis on agarose gel (1.5% Afllymetrix®) in running buffer 0.5x TBE (Invitrogen®) with the marker of 100bp (Invitrogen®) for 90 minutes at 100W of potence, 80 V of voltage and electric current of 80 A. The gels were stained with SYBR® Safe DNA gel stain solution (Invitrogen®) and visualized under UV light transilluminator (Loccus Biotecnologia).
The preparation of PCR consisted of 20 picomoles of primer C1 and C4, 40 picomoles of primers pg3 and pg50 (Table 1), 10 mM of Tris-HCl, 50 mM of KCl, 200 μM of each deoxynucleotide triphosphates (DNTPs), 5.5 mM of MgCl2 and 1,25 U of Taq DNA polymerase and 20 ng of DNA (Invitrogen®). The amplification was according to the following steps: 1 initial denaturation cycle at 94 °C for 4 minutes; 25 cycles of: denaturation at 94 °C for 1 minute, annealing at 47 °C for 1 minute and extension at 72 ºC for 1 minute, followed by a final extension cycle at 72 ºC for 7 minutes (HARMON; RAMSOM; WESLEY, 1997) (link).
The amplified products (8μL) were submitted to electrophoresis on agarose gel (1.5% Afllymetrix®) in running buffer 0.5x TBE (Invitrogen®) with the marker of 100bp (Invitrogen®) for 90 minutes at 100W of potence, 80 V of voltage and electric current of 80 A. The gels were stained with SYBR® Safe DNA gel stain solution (Invitrogen®) and visualized under UV light transilluminator (Loccus Biotecnologia).
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