To confirm large aggregate/fibril formation, SDD-AGE was employed. Standard agarose (1.5%) was melted in a 1X TAE buffer (Fisher scientific) combined with 0.1% SDS, 0.5 μM 2,2,2-Trichloroethanol (Alfa Aesar) for stain-free detection and 1 μM Thioflavin T for fibril detection (Sigma). The hot solution was poured into an Owl EasyCast B1A mini gel system (Thermo Fisher Scientific). NANOG WR domain and CTD gel samples were first formed by diluting stock samples in 7 M GdnHCl to PBS buffer. The gel/precipitates were resuspended in 6X sample buffer (5:1) and pipetted for several times before 5 min RT incubation. Electrophoresis was performed in 1X TAE containing 0.1% SDS for 90 min at 80 V. After electrophoresis, the gel was imaged using the stain-free and Alexa 488 filters on the ChemiDoc MP imaging system (Bio-Rad). 3 and 10 μg BSA were used as negative controls.
Tae buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
TAE buffer is a commonly used buffer solution for electrophoresis applications. It is primarily used to maintain the pH and ionic environment during the separation of nucleic acids, such as DNA and RNA, in agarose gel electrophoresis.
Automatically generated - may contain errors
Lab products found in correlation
Genelute pcr clean up kit, by Merck Group (2 mentions)
Phusion hot start 2 high fidelity polymerase, by Thermo Fisher Scientific (2 mentions)
Sigma genelute plasmid kit, by Merck Group (2 mentions)
Dreamtaq polymerase, by Thermo Fisher Scientific (2 mentions)
Zymoclean gel dna recovery kit, by Zymo Research (2 mentions)
1 kb dna ladder, by Thermo Fisher Scientific (2 mentions)
Ethidium bromide, by Merck Group (2 mentions)
Mgcl2, by Merck Group (2 mentions)
Thermomixer, by Eppendorf (2 mentions)
Tween 20, by Merck Group (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
2 2 2 trichloroethanol, by Thermo Fisher Scientific (1 mentions)
Owl easycast b1a mini gel system, by Thermo Fisher Scientific (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Gotaq dna polymerase, by Promega (1 mentions)
Gotaq flexi buffer, by Promega (1 mentions)
1 kb dna ladder, by Promega (1 mentions)
Genesnap, by Syngene (1 mentions)
Mgcl2, by Promega (1 mentions)
Gotaq green master 2 ready mix, by Promega (1 mentions)
23 protocols using tae buffer
1
SDD-AGE for Aggregates and Fibrils Detection
2
RAPD-PCR based genetic analysis
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DNA was extracted from each isolate using the phenol/chloroform method and randomly amplified polymorphic DNA-PCR (RAPD-PCR) employed to estimate the genetic variations of the isolates. The reaction mixture (25 μL) comprised 5 μL of 5x GoTaq Flexi buffer (Promega, UK), 2.5 μL of dNTPs (0.4 mmol/L of each of dATP, dCTP, dGTP, dTTP; Promega), 1.5 μL of MgCl 2 (25 mmol/L; Promega), 1 μL of primer OPA-09 (5'-GGGTAACGCC-3'; 20 pmol/mL; Sigma Genosys, UK), 1 μL of GoTaq DNA polymerase (1.5 U/ μL; Promega), 1 μL of template DNA (5 ng/μL) and 13 μL of sterile water.
PCR was performed using Prime thermal cycler (with heated lid, 100 ºC; Techne) programmed for 40 cycles of denaturation (30 sec at 94 ºC), annealing (60 sec at 38 ºC) and extension (2 min at 72 ºC), with a final 10 min extension step (72 ºC). The reaction products were separated via agarose gel electrophoresis (1.5% in 1x TAE buffer [Fisher, UK] containing ethidium bromide [0.5 ng/mL]), using 1 Kb DNA ladder (Promega) as a molecular size indicator. DNA fragment patterns were visualized under UV light (Genesnap, Syngene) and analysed by Gel Compar II software.
PCR was performed using Prime thermal cycler (with heated lid, 100 ºC; Techne) programmed for 40 cycles of denaturation (30 sec at 94 ºC), annealing (60 sec at 38 ºC) and extension (2 min at 72 ºC), with a final 10 min extension step (72 ºC). The reaction products were separated via agarose gel electrophoresis (1.5% in 1x TAE buffer [Fisher, UK] containing ethidium bromide [0.5 ng/mL]), using 1 Kb DNA ladder (Promega) as a molecular size indicator. DNA fragment patterns were visualized under UV light (Genesnap, Syngene) and analysed by Gel Compar II software.
3
Molecular Typing of Klebsiella Virulence
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PCR assays were performed to determine the capsular serotypes for K1 or K2 and another 11 virulence encoding genes, including iucA, entB, uge, magA, terW, iroB, kfuB, rmpA, rmpA2, wabG, and irp2. PCR reaction mixtures were prepared in total volumes of 20 μL. Each reaction contained 1 μL of template DNA, 1 μL (equivalent to 10 pmol concentration) of each primer, and 10 μL of GoTaq® Green Master 2 × Ready Mix (Promega, Madison, WI, USA). The volume was completed to 20 μL by 7 μL of nuclease-free water. The PCR amplification conditions were as follows: initial denaturation for 5 min at 95 °C, then 35 cycles of denaturing at 95 °C for 30 s, annealing for 30 s and extension at 72 °C, followed by a final extension at 72 °C for 7 min. The appropriate annealing temperature for each pair of primers and the time for the extension step for each PCR amplicon are mentioned in Table S1 . DNA fragments of PCR products were detected through TAE agarose gel (1 %) (Bioline, London, UK) electrophoresis in 1 × TAE buffer (Thermo Scientific, Waltham, MA, USA). The Gene Ruler 1 kb DNA molecular weight marker (Thermo Scientific, Waltham, MA, USA) was used for sizing the PCR products.
4
HAdV-5 Infection in siRNA-Transfected HeLa Cells
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HeLa cells were transfected with indicated siRNAs for 36 h, followed by HAdV-5 infection for an additional 24 h. Total RNA was isolated using TRIreagent (Sigma), DNaseI-treated (Thermo Fisher Scientific), and subjected to TVN-PAT and ePAT analysis essentially as described (43 (link)). Gene-specific and universal primers for TVN-PAT and ePAT reactions are described in Table S1 . Semi-quantitative PCR amplifications were done using Phire Taq DNA polymerase (Thermo Fisher Scientific). PCR amplicons were separated on 2% Ultrapure agarose in 1× TAE buffer (Thermo Fisher Scientific) and were visualized with GelRed (Biotium).
5
Gel Electrophoresis Protocol for DNA Separation
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For gel electrophoresis,
400 μL SYBR Safe DNA Gel Stain, DNA loading dye, SDS solution
(6×), TAE buffer (Tris–acetate–EDTA) (50×),
TrackIt 100 bp DNA Ladder, and Ultra Low Range DNA Ladder were purchased
from Thermo Fisher Scientific. Agarose was purchased from Bio-Rad
Laboratories. NuPAGE 4–12%, Bis–Tris, 1.0 mm, and Mini
Protein Gel (12-well) were purchased from Invitrogen.
400 μL SYBR Safe DNA Gel Stain, DNA loading dye, SDS solution
(6×), TAE buffer (Tris–acetate–EDTA) (50×),
TrackIt 100 bp DNA Ladder, and Ultra Low Range DNA Ladder were purchased
from Thermo Fisher Scientific. Agarose was purchased from Bio-Rad
Laboratories. NuPAGE 4–12%, Bis–Tris, 1.0 mm, and Mini
Protein Gel (12-well) were purchased from Invitrogen.
6
High-Molecular-Weight DNA Extraction
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The molecular weight of the extracted DNA was determined by running 2 μL of each sample in 0.8% agarose gel, 1× TAE buffer (Thermo Fisher Scientific) for 4 h at 60 V. The DNA was stained with 1× SYBR safe (Invitrogen, Waltham, Massachusetts, USA). The molecular weight of the genomic DNA was estimated by comparison with a HMW DNA ladder (Lambda DNA/EcoRI+HindIII; Thermo Fisher Scientific). Digital images of the gels were generated in an Amersham 600 RGB imager (GE Healthcare, Chicago, Illinois, USA) using automatic collection parameters. The images of the gels were enhanced (contrast, homogenization, and background removal) in ImageJ version 1.52a (Rasband, 2018 ) prior to the digital analysis. The 1D gel electrophoresis image analysis software GelAnalyzer2010a (http://www.gelanalyzer.com ) was used to create profiles of the distribution of DNA fragments (Fig. 2 A, B) using the Lambda DNA/EcoRI+HindIII DNA ladder as a size reference.
For all species, HMW DNA was exclusively observed in the DNA samples extracted from cells homogenized using the LN2 grinding method. The DNA extracted using this treatment was observed as a tight, clear band over the 21.2‐kbp marker band, whereas DNA extracted from cells homogenized using the other treatments displayed substantial smearing and lacked a clear HMW DNA band, consistent with high DNA fragmentation (Fig.2 ).
For all species, HMW DNA was exclusively observed in the DNA samples extracted from cells homogenized using the LN2 grinding method. The DNA extracted using this treatment was observed as a tight, clear band over the 21.2‐kbp marker band, whereas DNA extracted from cells homogenized using the other treatments displayed substantial smearing and lacked a clear HMW DNA band, consistent with high DNA fragmentation (Fig.
7
Cascade Binding Affinity Assay
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To estimate binding affinity for Cascade complexes, 0.01 nM of 32P-5′-end-labelled SP1-CC-2 (target) and SP3-AA-2 (non-target) DNA fragments in the B buffer were incubated at room temperature for 10 min with increasing concentration of Cascade complexes. The samples were separated on 8% (w/v) polyacrylamide gel prepared in 1x TAE buffer (Thermo Fisher) for 2 h 30 min at 5 V/cm and visualised using an FLA-5100 phosphorimager (Fujifilm). Kd values were calculated as previously described by Tamulaitis et al. [63 (link)] from three independent experiments (see Additional file 14 for individual data values).
8
Molecular Cloning and DNA Analysis
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DNA fragments were amplified by PCR amplification with Phusion Hot Start II High Fidelity Polymerase (Thermo Scientific) and desalted or PAGE-purified oligonucleotide primers (Sigma-Aldrich, St. Louis, MO) performed according to the manufacturers’ instructions. Diagnostic PCRs were run with DreamTaq polymerase (Thermo Scientific). oligonucleotide primers used in this study are listed in Additional file 1 . PCR products were separated by electrophoresis on 1% (w/v) agarose gels (Thermo Scientific) in TAE buffer (Thermo Scientific) and, if required, purified with a Zymoclean Gel DNA Recovery kit (Zymo Research, Irvine, CA) or a GenElute PCR Clean-Up kit (Sigma-Aldrich). Yeast or E. coli plasmids were isolated with a Zymoprep Yeast Plasmid Miniprep II kit (Zymo Research), or a Sigma GenElute Plasmid kit (Sigma-Aldrich), respectively. A YeaStar Genomic DNA kit (Zymo Research) or an SDS/lithium acetate protocol [39 (link)] was used to isolate yeast genomic DNA. Yeast strains were transformed using the lithium acetate/polyethylene glycol method [40 (link)]. Single-colony isolates were obtained from three consecutive re-streaks on selective solid agar plates, followed by analytical PCR analysis of the relevant genotype. E. coli DH5α cultures were transformed by chemical transformation [41 (link)]. After isolation, plasmids were verified by restriction analysis and analytical PCR.
9
Optimized DNA Extraction for PCR-free Sequencing
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Genomic DNA was extracted from 80 mg of fresh leaves using a modified DNeasy Plant Mini Kit (Qiagen), adapted to produce high-quality and quantity DNA for PCR-free sequencing. The main modified steps were the addition of sodium bisulfite and RNAse A in the lysis buffer and an additional ethanol wash step before the DNA elution step. Integrity of genomic DNA was determined by running 2 μl of each sample in 1% agarose gel, 1X TAE buffer (Thermo Fisher Scientific) for 30 min at 100 V. The DNA was stained with 1X ethidium bromide solution (Sigma-Aldrich). The molecular weight of the genomic DNA was estimated by comparison with a 1-kb DNA ladder (Invitrogen). After this step, genomic DNA was checked for quality on NanoDrop ND8000 (Thermo Fisher Scientific) and quantified on Qubit (Thermo Fisher Scientific) by Picogreen dosage. Minimum conditions required for the preparation of PCR-free libraries were as follows: a 260/230 nm ratio over 1.8, a 260/280 nm ratio over 1.8, and at least 3 μg of genomic DNA. Illumina TruSeq PCR-free libraries were carried out using the DNA Shearing M220 (Covaris) and were checked by the Fragment Analyzer (Agilent). Sequencing was performed as 150-bp paired-end reads on NovaSeq6000 (Illumina). Both library preparation and sequencing were done by GeT-PlaGe platform, Genotoul, France.
10
Characterizing QD-Peptide Biosensor Conjugates
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Streptavidin-coated QD605ITK or QD655ITK (2 μM stock solution, Thermo Fisher, USA) and peptides were diluted into 10 mM 2-[4-(2-hydroxyethyl)-piperazin-1-yl]ethanesulfonic acid (HEPES, Calbiochem, USA) buffer (pH = 7.5). The final solutions had 10 nM QDs, various amount of peptides, and 2.4 M urea when indicated. After incubation for 1 hr at room temperature, glycerol (100%, Macron Fine Chemicals, USA) was added to each sample with a final concentration of 5% (v/v). The QD-biosensor conjugates were then loaded to a 10 cm long 1% (w/v) agarose (Invitrogen, USA) gels (4 μL sample per well) in 1 × TAE buffer (Thermo Fisher Scientific, USA), and run for 60 min at 100 V using BioRad PowerPac Basic power supply. Gels were then imaged using a Gel Logic 112 imaging system (Carestream, USA).
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