Molecular characterization was also performed by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) as an alternative molecular approach. The partial CAL gene was amplified using the primers CL1 and CL2A [42 (link)] as described above and digested with HhaI as described elsewhere [29 (link)]. Digested products were electrophoresed on 2.5% (w/v) agarose gels for 90 min at 100 V in the presence of GelRedTM (Biotium, Hayward, CA, USA). We included a lane loaded with 100-bp DNA Step Ladder (Promega, Madison, WI, USA), as well as one positive control from each of the following reference strains: S. brasiliensis (CBS 120339), S. schenckii (CBS 359.36), S. globosa (CBS 120340), and S. mexicana (CBS 120341). The bands were visualized using the L-Pix Touch (Loccus Biotecnologia, São Paulo, Brazil) imaging system under UV illumination.
100 bp dna step ladder
Manufactured by Promega
Sourced in United States
The 100 bp DNA Step Ladder is a molecular weight marker used to estimate the size of DNA fragments in agarose gel electrophoresis. It consists of a series of DNA fragments ranging from 100 to 1,000 base pairs in 100 base pair increments. This product can be used to determine the approximate size of unknown DNA samples by comparing their migration relative to the known fragment sizes in the ladder.
Automatically generated - may contain errors
Lab products found in correlation
Gelredtm, by Biotium (1 mentions)
Taqman universal master mix, by Roche (1 mentions)
Transcriptor high fidelity reverse transcription kit, by Roche (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Image master vds, by GE Healthcare (1 mentions)
Taq polymerase, by Promega (1 mentions)
4 protocols using 100 bp dna step ladder
1
PCR-RFLP Molecular Characterization of Sporothrix
2
Transcriptional Profiling of Oligodendrocytes
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Total RNA of Oli-neu cells and primary OPCs were extracted using either the RNeasy Mini Kit or miRNeasy Mini Kit (both Qiagen). Reverse transcription of mRNAs was performed with the Transcriptor High Fidelity Reverse Transcription Kit (Roche Applied Science). SncRNA715 was reverse transcribed by the TaqMan MicroRNA Reverse Transcription Kit with stem-loop RT primers specific for sncRNA715 sequence (Applied Biosystems) and amplified with the Taqman Universal Master Mix (Roche Applied Science) with specific primers and probes for sncRNA715 (Applied Biosystems). RT-PCR primers specific for rat MBP: 5′-AACATTGTGACACCTCGAACA-3′ and 5′-TGTCTCTTCCTCCCCAGCTA-3′. PCR of Ago1-4 mRNAs was carried out using the Pfu-DNA Polymerase according to manufacturer’s protocol. Primers were specifically designed to differentiate the four different murine Ago proteins (Ago1: 5′-AGCGGCAGGTGCCTAC-3′ and 5′-GGATCTGGCCACTGACC3-′, Ago2: 5′-ATATGCC TTCAAACCTCCACC-3′ and 5′-CCAGGGCCTGGATCGTC-3′, Ago3: 5′-ACAAGCCTGTCAGCACTAAC-3′ and 5′-CAGAC TTCTAGTGGCAGGTATG-3′, Ago4: 5′-GGAGCTCCTCTACA GCCAGG-3′ and 5′-CAAGCCGGGCATAATATGC-3′. Ago1-4 PCR products and 100 bp DNA Step Ladder (Promega) were separated on 1% agarose gels and stained with ethidium bromide (EtBr). RT-PCR products of Mbp mRNA and sncRNA715 were separated on 4% agarose gels and stained with EtBr.
3
Multiplex PCR for Staphylococcus aureus Toxin Genes
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PCR was used to detect the nuc gene and the SEs sea, seb, sec, sed, and see the specific primers described in Table-1. The PCR mixture contained 1.5 mM MgCl2, 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.1% Triton ® X-100, 200 mM of each deoxynucleotide triphosphate, 0.2 mM of the individual primers, and 0.625 U Taq polymerase (all the reagents from Promega Corp., Madison, Wisconsin, USA). The amplification was accomplished using an automatic thermocycler T-1 (Biometra). The positive control was S. aureus reference strain (ATCC 29213; sea, seb, sec, sed, and see).
The PCR cycles consisted of pre-heating at 95°C for 10 m, denaturation at 94°C for 5 m, annealing at 55°C for 30 s, and extension at 72°C for 1.5 min. The amplification was performed for 35 cycles with a final extension step at 72°C for 3.5 min.
The PCR products were analyzed using electrophoresis in 1.5% agarose gel containing 0.5 mg of ethidium bromide per mL. Gels were visualized and photographed with Image Master VDS (Pharmacia Biotech). The sizes of the amplification products were estimated by comparison with a 100 bp DNA step ladder (Promega Corp.).
The antibiogram pattern of the isolated S. aureus to some antimicrobial agents was constructed using the disk diffusion method, as described by Clinical and Laboratory Standards Institute [26] .
The PCR cycles consisted of pre-heating at 95°C for 10 m, denaturation at 94°C for 5 m, annealing at 55°C for 30 s, and extension at 72°C for 1.5 min. The amplification was performed for 35 cycles with a final extension step at 72°C for 3.5 min.
The PCR products were analyzed using electrophoresis in 1.5% agarose gel containing 0.5 mg of ethidium bromide per mL. Gels were visualized and photographed with Image Master VDS (Pharmacia Biotech). The sizes of the amplification products were estimated by comparison with a 100 bp DNA step ladder (Promega Corp.).
The antibiogram pattern of the isolated S. aureus to some antimicrobial agents was constructed using the disk diffusion method, as described by Clinical and Laboratory Standards Institute [26] .
4
RAPD-Based Binary Matrix Analysis
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Based on the RAPD pattern, binary matrices were constructed in which intense, reproducible bands were scored as 1 and the absence of bands was scored as 0. The 100 bp DNA Step Ladder (Promega) and the RAPD pattern of strains LMG 4252, LMG 6133 and LMG 8820 were used as references. The matrices were created with the help of PyElph 1.4 software [15] and were assembled in Microsoft Excel. An UPGMA (Unweighted Pair Group Method with Arithmetic Mean [21] ) dendrogram was constructed based on a Jaccard's similarity coefficient into FreeTree 0.9.1.50 [6] . Statistical analyses were conducted in FAMD 1.3 (Fingerprint Analysis with Missing Data) software [19] .
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