Fresh overnight cultures were used to prepare agar blocks for pulsed field gel electrophoresis (PFGE). Bacterial mass was suspended into cold 100 mM EDTA to achieve density of 8.5 McFarland units (Den-1B McFarland Densitometer, Grant-bio, Grant Instruments Ltd., Cambridgeshire, UK), followed by heating in +75 °C for 10 min [28 (link)]. After this the PulseNet Escherichia coli O157 PFGE protocol [29 (link)] with digestion of XbaI enzyme (New England Biolabs Inc., MA, USA) was followed. Separation of DNA fragments was done by using Chef DR III system (Bio-Rad Laboratories Inc., CA, USA). The fragments were visualized by SYBR Safe DNA staining (Thermo Fisher Scientific, Waltham, MA, USA) and imaged with AlphaImager HP (Alpha Innotech, Genetic Technologies Inc., FL, USA). PFGE patterns were examined using GelComparII software (version 6.6 Applied Maths NV, Belgium) to perform UPGMA (unweighted pair-group method using arithmetic average clustering) based analysis with the Dice similarity coefficient. Similarity cut-off was 85% to separate clusters and optimization and position tolerance were both set at 1.5%.
Sybr safe dna staining
Manufactured by Thermo Fisher Scientific
Sourced in United States
SYBR Safe DNA staining is a fluorescent dye used for detecting and visualizing DNA in agarose gels. It binds to double-stranded DNA and emits green fluorescence upon excitation with blue light.
Automatically generated - may contain errors
Lab products found in correlation
Xbai enzyme, by New England Biolabs (1 mentions)
Alphaimager hp, by Bio-Techne (1 mentions)
Chef dr 3 system, by Bio-Rad (1 mentions)
Gel doc 2000, by Bio-Rad (1 mentions)
Gotaq green master mix, by Promega (1 mentions)
Thermal cycler, by Bio-Rad (1 mentions)
Omniscript reverse transcriptase kit, by Qiagen (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Gotaq long pcr master mix, by Promega (1 mentions)
2 ml tube, by Eppendorf (1 mentions)
Dneasy plant mini kit, by Qiagen (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
4 protocols using sybr safe dna staining
1
PFGE-based Molecular Typing for E. coli O157
2
Malaria Plasmodium SSU rRNA Amplification
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A partial small subunit ribosomal RNA (SSU rRNA) gene was the target gene for polymerase chain reaction (PCR) amplification of malaria Plasmodium. The primers used in this PCR were adapted from Singh et al. [5 (link)]; rPLU1 and rPLU5 targeted to SSU rRNA resulted in Plasmodium spp. 1640 bp amplicons. PCR amplification was performed using a thermocycler machine (Gene Amp® PCR System 9700, Applied Bio SystemTM, Foster City, CA, USA). Each reaction volume of 25 μL contained 1 μL of 10 pmol/μL of each primer (Integrated DNA Technologies, Singapore), 12.5 μL of Go Taq® Green Master Mix (Promega, Madison, WI, USA), and 2.5 μL of 25–50 ng/μL of DNA templates and was adjusted with nuclease-free water to the total volume of 25 μL. The PCR process was run under the following conditions: pre-denaturation at 94°C for 3 min; 40 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 30 s; and a final extension at 72°C for 5 min. The PCR products were visualized using 1.8% agarose gel electrophoresis, stained with SYBR Safe DNA Staining (Thermo Scientific, USA), and detected using a Gel Doc 2000 (Bio-Rad, USA) [5 (link), 6 (link)].
3
Reverse Transcription-PCR Protocol
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Total RNA was isolated from cells using Trizol reagent according to the manufacturer’s instructions (Invitrogen, Burlington, ON, Canada). After reverse transcription with an Omniscript reverse transcriptase kit from QIAGEN (Toronto, ON, Canada), cDNA was amplified using the following conditions: 94°C for 1 min, followed by 30–40 cycles of the following: 94°C for 1 min, 58–64°C for 1min (depending on the primers) for hybridization temperature, and 72°C for 1 min, followed by a final extension step at 72°C for 10 min. PCR assays using equal amounts of RNAs that were reverse-transcribed and amplified by PCR with genes specific primers (Table 1
4
Genotyping Leaf Tissue for LanFTc1 Alleles
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Young leaf tissue from three biological replicates per plant was sampled. Frozen (−80°C) plant tissue (50 mg) was homogenized using TissueLyser II (Qiagen, Hilden, Germany) and two stainless steel beads (ø 5 mm) in 2 ml tubes (Eppendorf, Hamburg, Germany). DNA was isolated using DNeasy Plant Mini Kit (Qiagen). PCR was performed using GoTaq Long PCR Master Mix (Promega, Mannheim, Germany) and published LanFTc1_INDEL2 primers (Taylor et al., 2019 (link)), provided here for reference in Supplementary Table 2 . PCR conditions were as follows: initial denaturation (94°C for 2 min), then 35 cycles composed of denaturation (94°C for 30 s), annealing (62°C for 30 s), and elongation (72°C for 5 min), followed by the final extension (72°C for 10 min). Products were resolved by agarose gel electrophoresis and SYBR Safe DNA staining (Invitrogen, Carlsbad, CA, United States) and visualized on UV-transilluminator (Uvitec, Thermo Fisher Scientific, Waltham, MA, United States). Wild P27255 allele (ku, without deletion) was encoded as “A,” Palestinian allele (Pal, 1208 bp deletion) as “B,” 83A:476 allele (Ku, 1423 bp deletion) as “C,” and Krasnolistny allele (Jul, 5162 bp deletion) as “D.”
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