Genetic relatedness of the isolates was examined by PFGE in a CHEF-DRIII apparatus (Bio-Rad Laboratories, Hercules, and CA) following digestion of genomic DNA with XbaI enzyme (New England Biolabs, Massachusetts) according to Tenover et al. [20 (link)]. The PFGE images were processed and the dendrogram was calculated by FPQuest software v4.5 (Biorad laboratories inc, Hercules, California, USA.) using Dice coefficient and UPGMA (unweighted pair group method using arithmetic averages). Isolates having more than 95% similarity were considered identical.
Xbai enzyme
Manufactured by New England Biolabs
Sourced in United States
XbaI is a type II restriction endonuclease enzyme that recognizes and cleaves the palindromic DNA sequence 5'-TCTAGA-3'. It is commonly used in molecular biology for the digestion and analysis of DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Chef dr 3 system, by Bio-Rad (4 mentions)
Chef dr 3 apparatus, by Bio-Rad (2 mentions)
Fpquest software v4, by Bio-Rad (1 mentions)
Mung bean nuclease, by New England Biolabs (1 mentions)
Megascript t7, by Thermo Fisher Scientific (1 mentions)
Sybr safe dna staining, by Thermo Fisher Scientific (1 mentions)
Alphaimager hp, by Bio-Techne (1 mentions)
Chef dr 3, by Bio-Rad (1 mentions)
T7 rna polymerase, by Promega (1 mentions)
Anti hbx antibody, by Abcam (1 mentions)
Pcmv vector, by Beyotime (1 mentions)
Cell lysis buffer, by Beyotime (1 mentions)
T7 high yield rna synthesis kit, by New England Biolabs (1 mentions)
Genejet rna purification kit, by Thermo Fisher Scientific (1 mentions)
Vaccinia capping system, by New England Biolabs (1 mentions)
Fast mutagenesis kit, by Vazyme (1 mentions)
Mmessage mmachine sp6 kit, by Thermo Fisher Scientific (1 mentions)
Hiscribe t7 arca mrna kit, by New England Biolabs (1 mentions)
Chef dr 2 system, by Bio-Rad (1 mentions)
13 protocols using xbai enzyme
1
Genetic Relatedness Analysis by PFGE
2
Generating HCV Genotype 2a Virus
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The plasmid pJFH1, containing the full‐length genomic cDNA sequence of the HCV genotype 2a strain and the N17/JFH117 , 18 plasmid were linearized using XbaI enzyme (New England Biolabs) and then treated with Mung Bean Nuclease (NEB) prior purification. Linearized plasmids were used as a template to generate in vitro transcribed RNA using MEGAscript T7 (Life Technologies, Milan, Italy). The 10 μg of RNA were electroporated into Huh7 cells as previously described.19
3
PFGE-based Molecular Typing for E. coli O157
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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%.
4
Pulsed-field Gel Electrophoresis of Salmonella
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PFGE was performed according to the standard operating procedure for pulsenet PFGE of Salmonella serotypes (https://www.cdc.gov/pulsenet/pathogens/pfge.html ) with CHEF DRIII (Bio-Rad, USA). All isolates, including the reference strain-Salmonella serovar Braenderup H9812 in this study, were digested with XbaI enzyme (New England Biolabs, Leusden, The Netherlands). The cluster analysis of PFGE was performed using BioNumerics software (Version7.6; Applied Maths), and the Dice coefficient was determined using the unweighted pair group method with arithmetic averages (UPGMA). Band comparison was performed using dice coefficient with 1.50% optimization and 1.50% position tolerance. If the PFGE patterns of isolates have the same numbers of bands and the same apparent size, isolates are designated genetically indistinguishable[22 (link)].
5
Pulsed-field Gel Electrophoresis for Salmonella Typing
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PFGE was performed according to PulseNet protocol developed by the CDC [24 ]. S. enterica serovar Braenderup (ATCC BAA 664) was used as the control strain. Briefly, agarose plugs containing genomic DNA treated with Proteinase K Buffer were incubated with 20 units of XbaI enzyme at 37 °C for 2 h (New England BioLabs, Ipswich, MA, USA). DNA separation was performed with 1% SeaKem Gold agarose gels in 0.5 M Tris borate–EDTA buffer at 14 °C for 18 h with pulse times between 2.16 and 63.8 s using a CHEF DR III apparatus (Bio-Rad, Hercules, CA, USA). Gels were stained with ethidium bromide, visualized under UV light, and photographed. DNA fingerprints were analyzed using Bionumerics 7.1 software (Applied Maths, Austin, TX, USA). A phylogenetic tree was constructed using the Unweighted Pair Group Method, with Arithmetic Mean method with Dice coefficient at an optimization setting of 1% and a position tolerance setting of 1.5%, as recommended previously [25 (link)].
6
Molecular Typing of Multidrug-Resistant Enterobacteriaceae
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Clonal relationship of the isolates was evaluated by pulsed-field gel electrophoresis (PFGE) [15 (link)]. Total DNA from Escherichia coli, Klebsiella pneumoniae and Enterobacter spp. were digested using the XbaI enzyme (New England Biolabs, Ipswich, MA, USA). The generated fragments were separated by PFGE using a CHEF-DR III System (Bio-Rad) creating a unique PFGE profile for each clonal strain. Multilocus sequencing typing was performed for pan-drug aminoglycoside-resistant strains belonging to the species E. coli (strain no. 31) and K. pneumoniae (strain no. 62) [16 (link)]. Sequence types (STs) were investigated using the online databases (http://genomicepidemiology.org/ ).
7
Clonal Diversity of E. coli Isolates
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The clonal relationship of E. coli isolates was evaluated by pulsed-field gel electrophoresis (PFGE) analysis, as described previously [35 (link),38 (link)], and multi-locus sequence typing (MLST). Seven housekeeping genes were used for E. coli (adk, fumC, gyrB, icd, mdh, purA, and recA). MLST analyses were performed according to EnteroBase (http://mlst.warwick.ac.uk/mlst/dbs/ Ecoli). For PFGE, total DNA from E. coli isolates was digested using the XbaI enzyme (New England BioLabs, Ipswich, USA). Then, the generated fragments were separated by PFGE using a CHEF-DR III System (Bio-Rad, Cressier, Switzerland).
8
Synthesis of SARS-CoV-2 Positive Control
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SARS-CoV-2 positive template control for SARS-CoV-2 Nucleocapsid gene (N1 and N2) and ORF1ab gene (N3) were generated in two-steps. The single-stranded synthetic DNA fragments (Supplementary Table S3) were concatenated and amplified by overlap extension PCR (OE-PCR) [19 (link)] for cloning in pJET.2/blunt cloning vector (CloneJET PCR Cloning Kit, Cat. K1232) as per manufacturer's instruction. N1, N2 and N3 synthetic fragment cloned plasmids were linearized at 3′ end using XbaI enzyme (NEB, Cat. R0145S). In Vitro Transcription (IVT) reaction was set up using T7 RNA polymerase (Promega, Cat. 207B) with each gel-purified plasmid and incubated at 37 °C for 2 h as described previously [20 (link)]. The resultant mixture was subjected to DNase I treatment (rDNase I DNA-free kit, Cat. No. 2224G, Invitrogen BioServices India Pvt. Ltd.) for digestion of left-over plasmid DNA and purification was confirmed by denaturing RNA agarose gel electrophoresis.
9
Co-immunoprecipitation of YY1 and HBx
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pCMV-vector was bought from Beyotime technology (Shanghai, China). YY1 (HA tag in N terminal) and HBx (His tag in N terminal) sequences were constructed into pCMV-vectors through KpnI and XbaI enzyme (NEB, Ipswich, MA, USA) digestion. PCMV-HA-YY1 and pCMV-His-HBx vectors were co-transfected into Huh7 cells and co-IP assays were proceeded 2 days later. Briefly, 500μl cell lysis buffer (Beyotime, Shanghai, China) was used and then 1:50 diluted anti-HA (CST 3724, Danvers, MA, USA) or anti-His antibody (CST 12698, Danvers, MA, USA) was added into the solution at 4°C for overnight. Then magnetic beads were added and incubated for another 2 h. Later, beads were collected by magnetic separation rack and washed by lysis buffer mentioned above for five times. And finally, proteins were received by adding sample buffer to the beads with blending and incubating at 100°C for 30 min. The collected proteins were detected through western blots by anti-YY1 (CST 63227, Danvers, MA, USA) or anti-HBx antibody (Abcam ab2741, Cambridge, UK) in a concentration of 1:1000 dilution.
10
Cas9 mRNA Synthesis and Purification
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Plasmids carrying Cas9, as previously described [8 (link)], were digested using the XbaI enzyme (New England Biolabs) and incubated for 3 h at 37 °C. The digested plasmids were recovered and purified. In vitro transcription was performed using the T7 high yield RNA Synthesis Kit (New England Biolabs), followed by RNA purification with the GeneJET RNA Purification Kit (Thermo). After purification, the 5’ end of the synthesized Cas9 mRNA was capped using the Vaccinia Capping System (New England Biolabs). The concentration of the capped product was determined after purification and stored at −80 °C for use.
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