100 bp dna ladder

With minor modifications, Listeria speciation was conducted according to the PCR method by Ryu et al. [45 (link)]. The multiplex PCR was designed for the speciation of Listeria species into L. grayi, L. innocua, L. ivanovii, L. monocytogenes, L. seeligeri, and L. welshimeri. Each 25 µL reaction contained 10 µL 2X Red Taq Mastermix (Ampliqon A/S, Odense, Denmark), 4 µL of 20 mM primer mix, 4 µL DNA template, and 7 µL of PCR water. The following thermocycler conditions were used: denaturation at 94 °C for 5 min, 35 cycles of denaturation at 94 °C for the 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 32 s, and final extension at 72 °C for 5 min. L. monocytogenes ATCC 19111 strain, L. grayi ATCC 19120 strain, L innocua ATCC 11288 strain, L. ivanovii ATCC 19119 strain, L. seeligeri ATCC 35967 strain, and L. welshimeri ATCC 35897 strain were used as a positive control, and E. coli strain ATCC 2922 served as a negative control, and water was used as blank. The amplicons were subjected to gel electrophoresis using 1% agarose gel containing ethidium bromide and 100 V for 1 h 45 min. A 100 bp DNA ladder (Thermo Fischer Scientific; Waltham, MA, USA) was used to estimate amplicon sizes. Post electrophoresis, the gel was visualized under ultraviolet light, and the image was captured using a gel documentation system (BIORAD; Hercules, CA, USA).

Genomic DNA was isolated from bulk cells transfected with CRISPR/Cas9 plasmids using the epicentre QuickExtract DNA Extraction Solution (Lucigen) according to the manufacturer’s protocol. PCRs were amplified from genomic DNA using primers RU-O-22929 and RU-O-24042 for U4.4 cells (full-length PCR product = 353 bp, digested =  ~ 330 bp +  ~ 23 bp); primers RU-O-22776 and RU-O-22777 were used for Aag2 cells (full-length PCR product = 412 bp, digested =  ~ 179 bp +  ~ 231 bp). PCRs were screened for editing efficiency using the Surveyor Mutation Detection Kit (IDT) according to the manufacturer’s instructions, and treated amplicons were visualized on ~ 1% agarose gels with 100 bp DNA ladder (NEB) and SYBR Gold (Thermo Fisher Scientific).

Five discriminatory loci (VNTR-4, VNTR-7, VNTR-10, VNTR-Lb4 and VNTR-Lb5) were used to characterize the isolates and the extracted DNA samples as described by Salaün and colleagues in 2006 [21 (link)]. Briefly, 5 µL of boiled isolate or extracted DNA was added to 20 µL of the KAPA2G Robust HotStart PCR kit (Kapa Biosystems Resnova) reaction mixture that contained 10 pmol of each primer. The PCR was carried out as follows: 95 °C for 15 min and 35 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min. An additional extension for 10 min at 72 °C was added to the end of the run. The PCR products were analyzed on 2% agarose gels stained with EuroSafe Nucleic Acid Stain (Euroclone), and the molecular weights were estimated by comparison with a 100-bp DNA ladder (Thermo Fisher Scientific). If the length of PCR product was unclear, we sequenced it using the same sequencing protocol described for the MLST analysis.

Total RNA was extracted from a pool of wild-type zebrafish embryos at 24 hpf, 2 and 3 dpf, and from adult organs (heart, liver eye, ovary/testis, brain, and skeletal muscle) using Qiazol (Qiagen) according to manufacturer’s instructions. For removal of residual DNA, the extracted RNA was incubated with DNAse I (Sigma-Aldrich) for 15 min at room temperature. A total of 2 µg of total RNA was reverse-transcribed using the High-Capacity RNA-to-cDNA Kit (Applied Biosystems, Waltham, MA, USA) and used as template for RT-PCR analysis with MangoTaq DNA Polymerase (Bioline) and eef1a1l1 (NM_131263.1) as reference gene. Primers for tmem43 and eef1a1l1 were tmem43-WISH_for: 5′-CGGAAGTGGAGTTAGCGTTAGTG-3′ and tmem43-WISH_rev: 5′-GCATGCACAGACACACTGTAG-3′, with an amplicon size of 357 bp; eef1a1l1_for: 5′-GCCCCTGGACACAGAGACTTCATCA-3′ and eef1a1l1-_rev 5′-ATGGGGGCTCGGTGGAGTCCAT-3′, with an amplicon size of 211 bp. PCR products were agarose-gel separated, stained with Midori Green (Nippon Genetics Europe, Düren, Germany) and imaged under UV light using a PXi imaging system (Alpha Metrix Biotech, Rödermark, Germany). Amplicon size was compared with a 100 bp DNA ladder (Thermo Fisher Scientific).

The genomic DNA of seven chickpea genotypes was extracted from young leaves using the CTAB method [39 ]. Purified DNA was used for PCR amplification using ASR gene specific forward 5’-GGGAACTAATCCTTTCCAAACA-3’ and reverse 5’-CTGCAGCACCTAACTCACCA-3’ primer custom synthesized by G-Biosciences, USA [83 ]. PCR was carried out in the Chickpea Molecular Breeding Laboratory, Division of Genetics, ICAR-IARI using a G-STORM thermal cycler (Labtech, France). The PCR master mix comprised of 20ng of the template DNA, 1.6μl of 10X Tris borate-ethylenediamine-tetra acetic acid (TBE), 1μl of 10mM dNTP mix (Genei, Banglore), 1μl each of 5μM forward and reverse primer and 0.3μl of 3U Taq Polymerase (Genei, Banglore). The PCR cycling reaction consisted of three steps, starting with initial denaturation (90°C for 3min) followed by 38 cycles of denaturation (94°C for 20sec), annealing (55°C for 50sec) and elongation (72°C for 50sec) followed by final elongation (72°C for 7mins) [89 ]. The PCR amplicons were visualized on a 3% 1X TBE buffer using 100bp DNA ladder (Thermo Scientific, USA). The gel image was documented using UV light gel documentation system (UVITECH Imaging System, UK).

Blood samples were collected from patients and healthy volunteers in tubes containing ethylenediaminetetraacetic acid. Samples were stored at -20 °C until DNA isolation. DNA was isolated from whole blood using Pure Link Genomic DNA Purification Kit (Thermo Massachusetts, USA). Polymerase chain reaction was performed using 2 mM dNTPs (Thermo Scientific R0242), 10 pmol of each primer, 1.5 mM MgCl₂, and 1×polymerase chain reaction buffer containing (NH₄)2SO₄ and 2 U Taq DNA polymerase (Thermo Scientific 0402 Massachusetts, USA). Amplification was performed on an automated thermal cycler (Techne Flexigene, Cambridge, UK). A 100-bp DNA ladder (Thermo Scientific) was used as standard size for each gel lane. The gel was visualized under a UV visualizing system (Vilber Eberhardzell, Germany).

The assay was trialed on 10 known samples from three black rhinos, three white rhinos and four Indian rhinos (S1 Table). Different sample types were used to test the utility of the assay, including rhino horn, bone, hair, blood and tissue. The presence and length of multiplex PCR products were assessed using 2% TAE agarose gel electrophoresis stained with GelRed (Biotium), with the inclusion of a 100 bp DNA ladder (ThermoFisher). Positive control samples from known white rhino (M.47191), black rhino (M.46281) and Indian rhino (M.39431) from the Australian Museum Mammal Collection were run alongside the unknown sample on the agarose gel to provide an effective means to visualize the amplicon size and hence identify the species of the unknown samples. Representative positive amplifications were purified using ExoSap-IT and Sanger sequenced at the Australian Genome Research Facility (Sydney, Australia) to ensure target species have indeed been amplified and no cross species amplification had occurred. Sequences were compared to those generated from the available reference specimens and sequences available on GenBank to confirm correct species identification.