Amplification of the partial mtCO1 fragment was performed using forward primer 2195Bt (5ʹ-TGRTTTTTTGGTCATCCRGAAGT-3ʹ) and reverse primer C012/Bt-sh2 (5ʹ-TTTACTGCACTTTCTGCC-3ʹ) (Mugerwa et al. 2018 ). PCR reaction mixtures (20µL) contained 10µL of 2 × reSource™ Taq Mix (reSource Taq DNA Polymerase, 6 mM MgCl2, 2 mM dNTPs) (Source BioScience, UK), 1µL of each 10 µM primer, 6µL of molecular biology-grade water (Sigma-Aldrich) and 2µL of DNA template. Initial denaturation (94 °C 2 min) was followed by 35 cycles of denaturation (94 °C, 20 s), primer annealing (52 °C, 30 s) and extension (72 °C, 1 min). A final extension (72 °C, 10 min) was performed before storing reactions at 4 °C. Electrophoresis of PCR products was on 2%(w/v) agarose gels in 0.5 × TBE stained with RedSafe™ (iNtRON Biotechnology, Korea). PCR products were visualised under UV light (302 nm) and those of the expected size (864 bp) purified for sequencing and cloning using a reSource™ PCR purification kit (Source BioScience, UK). Purified PCR products were sent for Sanger sequencing (Source BioScience, UK). Where a novel sequence was identified, purified PCR products were cloned from three separate PCR reactions using the pGEM®-T easy vector kit (Promega, UK) and resequenced to confirm the novel sequence. Sequences generated were deposited in GenBank (accession numbers MK444227-MK445130).
Molecular biology grade water
Manufactured by Merck Group
Sourced in United States, United Kingdom
Molecular biology grade water is a highly purified water product designed for use in sensitive molecular biology applications. It undergoes extensive filtration and purification processes to remove impurities, ions, and organic contaminants, ensuring it meets stringent quality standards for use in critical laboratory procedures.
Automatically generated - may contain errors
Lab products found in correlation
Qubit fluorometer, by Thermo Fisher Scientific (4 mentions)
Dsdna assay kits, by Thermo Fisher Scientific (2 mentions)
Reverse primer 28s cl r, by Merck Group (2 mentions)
Ssdna oligonucleotide, by Integrated DNA Technologies (2 mentions)
Alexafluor 647 labeled ssdna oligonucleotides, by Integrated DNA Technologies (2 mentions)
Ultrapure equimolar deoxynucleotide triphosphate dntp sodium salt solution, by New England Biolabs (2 mentions)
Alexafluor 647 c2 maleimide free af, by Thermo Fisher Scientific (2 mentions)
Sodium chloride nacl magnesium chloride mgcl2, by Merck Group (2 mentions)
Noble agar, by Thermo Fisher Scientific (2 mentions)
Quantifluor r dsdna system a, by Promega (2 mentions)
Forward and reverse pe primers, by Illumina (2 mentions)
Nextseq high output 75 bp single end run, by Illumina (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Mx3005p rt pcr platform, by Agilent Technologies (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Sucrose, by Merck Group (2 mentions)
Sanger sequencing, by Source Bioscience (1 mentions)
Resource pcr purification kit, by Source Bioscience (1 mentions)
Pgem t easy vector kit, by Promega (1 mentions)
Redsafe, by iNtRON Biotechnology (1 mentions)
42 protocols using molecular biology grade water
1
Mitochondrial COI Amplification and Sequencing
2
Drug Injection Protocols for Behavioral Studies
3
Concentration of SARS-CoV-2 Viral RNA from Samples
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SARS-CoV-2 viral RNA was concentrated using a modified PEG (Polyethylene glycol) precipitation protocol (Medema et al., 2020 ; Wu et al., 2020 ). Thoroughly mixed 45 ml aliquots from each sample were centrifuged at 4500 g for 30 min at 4 °C (the centrifuge was previously cooled down to 4 °C). 40 ml of the supernatant were further added to fresh 50 ml falcon tubes containing 100 g/L PEG 8000 (Sigma Aldrich) and 22.5 g/L NaCl (Sigma Aldrich), followed by centrifugation at 12,000 g for 1 h 30 min at 4 °C. The supernatant was carefully removed and an additional centrifugation step was carried out in order to remove the remaining liquid (12,000 g, 5 min, 4 °C). The pellet was resuspended in 400 µl molecular biology grade water (Sigma Aldrich) and 400 µl CTAB (Hexadecyltrimethylammonium bromide; Promega) buffer. Prior to the RNA extraction, 40 µl of Proteinase K (Promega) were added to each tube and inverted carefully in order to obtain a homogenous suspension. The suspension was further transferred to a tube containing disruptor beads and run on FastPrep24 machine for 40 s at 6 m/s. Subsequently, 400 µl of each sample were used for further RNA extraction. The RNA extraction was performed on Maxwell® RSC AS4500 (Promega) device using Viral RNA/DNA Concentration and Extraction Kit from Wastewater (Promega). The SARS-CoV-2 viral RNA was eluted in 100 µl TE buffer.
4
Screening E. coli Isolates for tet(A) Alleles
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E. coli isolates that had not previously been sequenced were screened for presence of tet(A), and, if applicable, the nature of the tet(A) allele. Template for PCR reactions was prepared by pelleting 30 to 50 μl of overnight cultures, resuspending cells in an equal volume of molecular biology grade water (Sigma-Aldrich), and heating for 10 minutes at 98°C. PCR screening was performed using DreamTaq Green PCR Master Mix (Thermo Fisher Scientific), according to manufacturer’s protocol with 2 μl DNA template per reaction. Primers (Sigma-Aldrich) are described in S11 Table . For the multiplexed screen for tet(A) presence and tetR(A) allele, the protocol used was as follows (per reaction): 12.5 μl MasterMix, 1 μl of each primer (tetAscreen_P2, tetAscreen_P4, tetRdelscreen_short_P1, tetRdelscreen_short_P2, all at 10 mM concentration; see S11 Table ), 6.5 μl water, and 2 μl DNA template. Unless specified otherwise, the PCR program was as follows: 1 minute at 95°C; 34 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute; 7 minutes at 72°C followed by cooling to 4°C. PCR machines used were C1000 Touch Thermal Cycler (Bio-Rad) and GeneAmp PCR System 9700 (Applied Biosystems), interchangeably. PCR products were analysed by electrophoresis in 1% agarose gels, except the multiplex PCR products which were analysed using 2% agarose gels.
5
PCR Amplification with Platinum Taq DNA Polymerase
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Each PCR amplification with Platinum™ Taq DNA Polymerase (Platinum™ Taq) (Thermo Fisher Scientific, Waltham, MA) was carried out in a 25 μl of mixture containing Platinum™ Taq (10 units/μl) 0.125 μl, 10X PCR Buffer Mg 2.5 μl, MgCl2 (50 mM) 1 μl, dNTP (2.0 mM each) 2.5 μl, Template DNA 2 μl, forward primer (10 μm) 0.75 μl and reverse primer (10 μm) 0.75 μl for either primer set 18S-CL-F3 and 28S-CL-R or ITS-CL-F2 and 28S-CL-R, and molecular biology grade water (Sigma-Aldrich, St Louis, MO) 15.375 μl. The thermal cycling program was one cycle of 95°C for 3 min; 36 cycles of 95°C for 30 sec, 50°C for 45 sec, 72°C for 3 min; and final extension at 72°C for 7 min.
6
Optimized Bile DNA Extraction Protocol
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Total DNA extraction from 1-ml bile samples was performed following an optimized protocol based on a previously described method [19 (link)], with slight modifications. Briefly, samples were centrifuged at maximum speed at room temperature for 10 min and pellets re-suspended in 0.5 ml of extraction buffer consisting of 200 mM Tris-HCl pH 7.0, 25 mM EDTA, 250 mM NaCl, and the following enzymes: 20 mg/ml lysozyme (Merck, Darmstadt, Germany), 5 μg/ml of lysostaphin (Sigma-Aldrich, Saint Louis, MO, USA), and 40 U/ml mutanolysin (Sigma-Aldrich). Enzymatic lysis was performed for 1 h at 37 °C; after that, SDS was added to a final concentration of 0.5% (w/v), and mechanical disruption was performed in a FastPrep FP120 apparatus (Qbiogene, Carlsbad, CA, USA). The lysate solution was treated with proteinase K, 1.5 M NaCl as previously described [39 (link)] and extracted with phenol/chloroform. Precipitation of DNA was performed with 0.1 volumes of 3 M sodium acetate pH 5.2 and 2.5 volumes of cold ethanol. The DNA was then pelleted, washed with 70% ethanol, resuspended in 50 μl of molecular-biology grade water (Sigma-Aldrich), and stored at − 20 °C until use. DNA concentration and quality was determined in a BioTek Epoch™ spectrophotometer system (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and in parallel in a Qubit fluorometer with dsDNA assay kits (Thermo Fisher Scientific).
7
PCR Amplification with pfu and DreamTaq
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Each PCR amplification with pfu DNA polymerase (Agilent, Santa Clara, CA) alone or combined with DreamTaq™ was carried out in a 25 μl of mixture containing pfu (2.5 units/μl) 0.75 μl (or plus DreamTaq™ (5 units/μl) 0.125 μl), 10× Pfu reaction buffer, 10× PicoMaxx™ buffer, or 10× DreamTaq™ buffer 2.5 μl, dNTP (25 mM each) 0.2 μl, Template DNA 2 μl, forward primer 18S-CL-F3 (10 μm) 1.25 μl, reverse primer 28S-CL-R (10 μm) 1.25 μl, and molecular biology grade water (Sigma-Aldrich, St Louis, MO) 16.85 μl (or 16.725 μl). The thermal cycling program was one cycle of 95°C for 2 min; 36 cycles of 95°C for 30 sec, 55°C for 45 sec, 72°C for 5 min; and final extension at 72°C for 7 min.
8
Efficient CRISPR-Cas Array Insertion in Clostridium
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To assess efficiency of the putative PAMs, oligonucleotides were designed to match the sequence of spc 53 from the C. saccharoperbutylacetonicum N1-4(HMT) CRISPR-Cas array with a 5’ CCA, CCT or CCC (Table 1 ). When annealed the primer pairs give a 5’ blunt end and a 3’ HindIII compatible overhang (Table 1 , ref 1–6). 10 pmol of each primer were suspended in 100 µl molecular biology grade water (Sigma), and annealed: Primer mix was incubated at 95°C for 5 min and then gradually cooled, 1°C/min, from 70°C to 30°C. The final step was a temperature decrease from 30°C to 4°C over 5 min. Annealed primers were used as the ‘insert’ in a ligation reaction using pMTL83251 cut with SmaI and HindIII (or ZraI/HindIII for the no PAM control). Colonies were screened by PCR and sequenced before transforming into C. saccharoperbutylacetonicum N1-4(HMT).
9
Fluorescent DNA Oligonucleotide Synthesis
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ssDNA oligonucleotides and AlexaFluor™ 647-labeled ssDNA oligonucleotides were purchased from Integrated DNA Technologies Inc. Full oligonucleotide sequences can be found in the accompanying Supporting Information (SI) file. Ultrapure equimolar deoxynucleotide triphosphate (dNTP) sodium salt solution was purchased from New England Biolabs Inc. (Catalog #N0447S) as an equimolar mixture of 10 mM dATP, dCTP, dGTP and dTTP giving a final dNTP concentration of 40 mM. AlexaFluor™ 647 C2 maleimide (Free AF) was purchased from ThermoFisher Scientific (Catalog #A20347). All other chemicals including 97% 1, 1′-diethyl-2, 2′-cyanine iodide (PIC, CAS #:977-96-8); molecular biology-grade water; sodium chloride (NaCl); magnesium chloride (MgCl2); and TRIS (2-Amino-2-(hydroxymethyl)propane-1, 3-diol; pH 7.0) were sourced from Sigma-Aldrich.
10
Non-invasive RNAi Delivery in Varroa and Psoroptes Mites
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A non-invasive immersion technique for RNAi developed in Varroa destructor [23 (link)] was adapted for P. ovis using modifications [20 (link)]. Briefly, mites, or their eggs, were immersed in the detached cap of a 1.5 mL micro-centrifuge tube in 15 µL of a solution containing either a fluorophore-labelled AllStars™ Negative Control siRNA (Qiagen, UK) (“fluoro-siRNA”) in 0.9% w/v NaCl solution, final fluoro-siRNA concentration 0.05 µg/µL, or 15 µL 0.9% w/v NaCl solution only. The mites were incubated at 4 °C overnight then washed three times in molecular biology grade water (Sigma, UK) before photographic images were captured through an Axiovert 25 CFL inverted fluorescent microscope (Zeiss, Germany) with 10× Achrostigmat magnification lens (Zeiss, Germany) using a D90 AF-5 DX NIKKOR digital camera with 18–105 mm f/3.5–5.6 G ED VR lens kit (Nikon, Japan).
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