High-titer (1011–1012 pfu/ml in MgSO4 10 mM) lysates were obtained from each bacteriophage propagated in S. Typhimurium LB5000 strain (SGSC181; University of Calgary) and by ultracentrifugation at 51,000 × g for 2 h (OptimaTM L-80; Beckman, CA, USA) (Sambrook et al., 1989 ). Bacteriophage DNA was isolated using a phenol-chloroform method (Sambrook et al., 1989 ) with slight modifications. Phage suspensions were treated with DNase I (80 U/ml; Roche Diagnostics GmbH, Germany) and RNase I (80 μg/ml; Roche Diagnostics GmbH, Germany) at 37°C for 2 h. Following the addition of 0.5% sodium dodecyl sulfate (SDS, Sigma-Aldrich, St. Louis, MO, USA) and 200 μg proteinase K (Roche Diagnostics GmbH, Germany)/ml, they were incubated at 56°C for 2 h. Phage DNA was then extracted using phenol:chloroform and precipitated with ethanol. DNA integrity was checked by using a 0.7% agarose gel electrophoresis stained with Red Safe 1X (Intron Biotechnology; Seongnam-Si, Korea); the concentration was determined in a NanoDrop ND 1000 instrument (Thermo Scientific, DE, USA).
Red safe 1x
Manufactured by iNtRON Biotechnology
Red Safe 1X is a nucleic acid stain that can be used to detect DNA and RNA in agarose gels. It is a ready-to-use solution that can be directly added to the gel or used for post-staining. Red Safe 1X emits a red fluorescence when bound to nucleic acids and can be visualized using a UV or blue-light transilluminator.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions)
Rnase 1, by Roche (1 mentions)
Optima l 80, by Beckman Coulter (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Dnase 1, by Roche (1 mentions)
Proteinase k, by Roche (1 mentions)
2720 thermocycler, by Thermo Fisher Scientific (1 mentions)
Quantity one v452, by Bio-Rad (1 mentions)
Puretaq ready to gotm pcr beads, by GE Healthcare (1 mentions)
Abi prism 310 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Gel doc xr system, by Bio-Rad (1 mentions)
2 protocols using red safe 1x
1
High-Titer Bacteriophage DNA Isolation
2
PCR Amplification and Sequencing of Cytochrome b
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PCR reactions were performed in a final volume of 25 μL using PureTaq TM Ready-to-Go TM PCR beads (GE Healthcare UK Ltd) with the addition of molecular-grade water, primers (final concentration 0.2 µM) and 50 ng of DNA template. The thermal cycling parameters were as follows: 95°C for 5 min, followed by 25 cycles of 95°C for 20 s, 69°C for 30 s, 72°C for 30 s and a terminal extension step of 72°C for 5 min. Negative controls (molecular-grade water) were included in each set of reactions. These PCR reactions were carried out in an Applied Biosystems 2720 Thermocycler.
The amplification products were tested in a 2% agarose gel (Pronadisa), containing RedSafe™ 1X (iNtRON Biotechnology) in 0.5X TBE buffer (Sigma). DNA fragments were visualized using the Gel Doc XR System and the software Quantity One® v 4.5.2 (Bio-Rad).
Sequencing was used to verify the correct species assignation of the specimens used for the LFD method set-up. L14735 and H1549D (Kocher et al, 1989) were used for the amplification and subsequent sequencing on an ABI Prism 310 Genetic Analyzer (Applied Biosystems). The resulting Cytochrome b nucleotide sequences were analyzed using Megablast with those present in the NCBI database.
The amplification products were tested in a 2% agarose gel (Pronadisa), containing RedSafe™ 1X (iNtRON Biotechnology) in 0.5X TBE buffer (Sigma). DNA fragments were visualized using the Gel Doc XR System and the software Quantity One® v 4.5.2 (Bio-Rad).
Sequencing was used to verify the correct species assignation of the specimens used for the LFD method set-up. L14735 and H1549D (Kocher et al, 1989) were used for the amplification and subsequent sequencing on an ABI Prism 310 Genetic Analyzer (Applied Biosystems). The resulting Cytochrome b nucleotide sequences were analyzed using Megablast with those present in the NCBI database.
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