Cytidine deamination activity on ssDNA was assessed using the established PCR-based assay with a protein concentration adjusted to an overall product formation of ~10% activity to ensure assay sensitivity as described before26 (link). Deamination reactions (37 °C) were performed in a total volume of 10 μl in 25 mM Tris pH 7.0, 1ul protein, 0.1 μg/μl BSA and 1 pmol/μl ssDNA substrate containing CCC, TTC or TGC deamination motifs. The deamination reaction mixture (1ul) was amplified by PCR, incubated (1 h at 37 °C) with the StuI, DraI or XbaI restriction enzymes (New England Biolabs) respectively to cleave the deaminated product, which was resolved from the undeaminated (uncleaved) substrate on a polyacrylamide gel.
Xbai restriction enzyme
Manufactured by New England Biolabs
Sourced in United States, Canada
XbaI is a type II restriction enzyme that recognizes and cleaves the palindromic DNA sequence 5'-TCTAGA-3'. It is commonly used in molecular biology applications such as DNA cloning, mapping, and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Mmessage mmachine transcription kit, by Thermo Fisher Scientific (2 mentions)
Seakem gold agarose, by Lonza (2 mentions)
Hindiii, by New England Biolabs (2 mentions)
Gelred, by Biotium (2 mentions)
Seakem gold agarose, by Thermo Fisher Scientific (2 mentions)
Uv imager, by Bio-Rad (2 mentions)
Bamhi, by New England Biolabs (1 mentions)
Plasmid giga kit, by Qiagen (1 mentions)
Hindiii hf, by New England Biolabs (1 mentions)
Pvax1 plasmid vector, by Thermo Fisher Scientific (1 mentions)
Neb 10 beta competent e coli high efficiency, by New England Biolabs (1 mentions)
Chef mapper xa system, by Bio-Rad (1 mentions)
Seakem gold agarose, by Merck Group (1 mentions)
Tryptic soy broth (tsb), by BD (1 mentions)
Tryptic soy agar, by BD (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Chef mapper, by Bio-Rad (1 mentions)
Pgl3 promoter vector, by Promega (1 mentions)
Fugene 6, by Promega (1 mentions)
Dual glo luciferase assay system protocol, by Promega (1 mentions)
24 protocols using xbai restriction enzyme
1
Quantitative Cytidine Deamination Assay
2
Engineered Fluorescent Protein Constructs
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H2B:mRuby was generated from H2B:GFP, generously provided by G. Hickson (University of Montreal). GFP was replaced with mRuby using BamHI (New England Biolabs) and XbaI restriction enzymes (New England Biolabs), and the pcDNA3:mRuby2 plasmid was kindly provided by C. Brett (Concordia University). A stable HeLa mCherry:tubulin cell line was generated previously (van Oostende Triplet et al., 2014 (link)). pEGFP-N1:importin-β was obtained by Addgene, made by P. Lavia (plasmid #106941). GST:importin-β was made by cloning importin-β cDNA from the pEGFP-N1 vector into pGEX-4T using NcoI and NotI (New England Biolabs). The anillin constructs for mammalian cell expression (GFP-tagged) or protein expression (MBP or GST-tagged) were generated previously (Piekny and Glotzer, 2008 ; Frenette et al., 2012 (link)). The 850KK851-DE or 887KK888-DE (NLS mutant), A703E; E721A or A740E; E758A (RBD mutant; Sun et al., 2015 (link)), 837DFEINIE843-AFAINIA or 874DFEINIE880-AFAINIA (weak I/F mutant; Piekny and Glotzer, 2008 ), 735LL736-DD or 772LL773-DD (strong I/F mutant) and combinations of mutations were generated in the anillin constructs by quickchange PCR. The His:RhoA and Myc:Ect2 (C-terminus) constructs were generated previously (Yüce et al., 2005 (link); Frenette et al., 2012 (link)). All constructs were verified by sequencing.
3
Linearization and In Vitro Transcription of nAChR Subunit Plasmids
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Plasmid pMXT construct of human nAChR α7 and plasmid pSP64 construct of human nAChR α4 were linearized with BamHI, and plasmid pT7TS constructs of human nAChR α3, α9, α10, β2, and β4 were linearized with XbaI restriction enzymes (NEB, Ipswich, MA, USA).
Plasmid pcDNA3.1/Hygro(+) construct of rat nAChR α7 was linearized using XbaI, and plasmid pSGEM constructs of rat nAChR α9 and α10 were linearized using NheI restriction enzymes (Promega, Madison, WI, USA). All linearized plasmid constructs were subjected to in vitro cRNA transcription using SP6 (human nAChR α7 and α4) and T7 (human nAChR α3, α9, α10, β2, and β4, and rat nAChR α7, α9 and α10) mMessage mMachine® transcription kits (AMBION, Foster City, CA, USA).
Plasmid pcDNA3.1/Hygro(+) construct of rat nAChR α7 was linearized using XbaI, and plasmid pSGEM constructs of rat nAChR α9 and α10 were linearized using NheI restriction enzymes (Promega, Madison, WI, USA). All linearized plasmid constructs were subjected to in vitro cRNA transcription using SP6 (human nAChR α7 and α4) and T7 (human nAChR α3, α9, α10, β2, and β4, and rat nAChR α7, α9 and α10) mMessage mMachine® transcription kits (AMBION, Foster City, CA, USA).
4
Cloning and Amplification of Luciferase Plasmid
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mRNA-encoding firefly luciferase (mRNA-Luc) was purchased from TriLink Biotechnologies (San Diego, CA, USA) with the substitution of N1-Methylpseudouridine for uridine.
To generate the pDNA-encoding firefly luciferase gene (pVAX1-Luc), the Luc gene from the pcDNA3-Luc plasmid (Addgene, Watertown, MA, USA) was cloned into the pVAX1 plasmid vector (ThermoFisher, Ottawa, ON, Canada) using HindIII-HF and XbaI restriction enzymes (New England Biolabs, Whitby, ON, Canada). pVAX1-Luc was transformed into NEB® 10-beta Competent E. coli (High Efficiency) (New England Biolabs, Ipswich, MA, USA)according to the manufacturer’s protocol). Large-scale amplifications of pVAX1-Luc were generated using the EndoFree QIAGEN Plasmid Giga Kit (Montreal, QC, Canada) according to the manufacturer’s instructions.
To generate the pDNA-encoding firefly luciferase gene (pVAX1-Luc), the Luc gene from the pcDNA3-Luc plasmid (Addgene, Watertown, MA, USA) was cloned into the pVAX1 plasmid vector (ThermoFisher, Ottawa, ON, Canada) using HindIII-HF and XbaI restriction enzymes (New England Biolabs, Whitby, ON, Canada). pVAX1-Luc was transformed into NEB® 10-beta Competent E. coli (High Efficiency) (New England Biolabs, Ipswich, MA, USA)according to the manufacturer’s protocol). Large-scale amplifications of pVAX1-Luc were generated using the EndoFree QIAGEN Plasmid Giga Kit (Montreal, QC, Canada) according to the manufacturer’s instructions.
5
Human nAChR Subunit cRNA Synthesis
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Plasmid
pMXT construct of the
human α7 nAChR subunit was linearized with BamHI, and plasmid pT7TS constructs of human α3, α9, α10,
β2, and β4 nAChR subunits were linearized with XbaI restriction enzymes (NEB, Ipswich, MA) for in vitro
cRNA transcription using SP6 (hα7) and T7 (hα3, α9,
α10, β2, and β4) mMessage mMachine transcription
kits (AMBION, Foster City, CA).
pMXT construct of the
human α7 nAChR subunit was linearized with BamHI, and plasmid pT7TS constructs of human α3, α9, α10,
β2, and β4 nAChR subunits were linearized with XbaI restriction enzymes (NEB, Ipswich, MA) for in vitro
cRNA transcription using SP6 (hα7) and T7 (hα3, α9,
α10, β2, and β4) mMessage mMachine transcription
kits (AMBION, Foster City, CA).
6
PFGE Subtyping of Salmonella in PulseNet
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PFGE analysis was performed based on the standardized protocol for the subtyping of Salmonella in the PulseNet [32 (link)]. Purified DNA was digested using XbaI restriction enzyme (NEB) in a final volume of 100 μl and incubated at 37 °C for 3 h and embedded in a 1% SeaKem Gold Agarose (Sigma Aldrich) prepared using 0.5x TBE buffer. The reaction was run for 18 h using the Chef Mapper XA system (Bio-Rad) in order to resolve the DNA macro-restriction fragments. Salmonella enterica Typhimurium was used as a control. Macro-restriction patterns were compared using the FPQuest cluster analysis based on the Dice correlation coefficient, while dendograms were constructed using the unweighted-pair group method using average linkages UPGMA.
7
Genetic Relatedness of AR E. coli by PFGE
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The genetic relatedness among the AR E. coli isolates was determined by standard pulsed-field gel electrophoresis (PFGE) using CHEF MAPPER (Bio-Rad Laboratories, USA) according to the manufacturer's instructions. Briefly, AR E. coli isolates cultured overnight in Tryptic Soy Broth (Becton, Dickinson and Company) were streaked onto Tryptic Soy Agar (Becton, Dickinson and Company) and incubated at 37℃ for 14 to 18 h. The bacterial colonies of each isolate were then suspended in 0.8% saline and adjusted to 4.0 McFarland. Next, suspensions were embedded in 1.0% agarose plugs and lysed with proteinase K (Sigma-Aldrich, USA). The lysed plugs were then digested for 2 h with 50 U of XbaI restriction enzyme (New England Biolabs, USA) at 37℃. Digested plugs were subsequently placed on 1.0% SeaKem Gold agarose (Lonza, USA) and PFGE was carried out at 6.0 V for 19 h with a ramped pulse time of 6.76 to 35.38 sec in 0.5× Tris-Borate-EDTA (TBE) buffer at 14℃. BioNumerics software (Applied Maths, Belgium) was used to establish a DNA similarity matrix using the dice coefficient (0.5% optimization, 1.0% tolerance) and the un-weighted pair group method.
8
MFF 3'UTR Luciferase Assay
9
Genomic Fingerprinting of E. coli Isolates
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Pulsed-field gel electrophoresis (PFGE) was performed with the XbaI restriction enzyme (New England Biolabs, Beverly, MA, USA) as described previously.34 (link) PFGE profiles were analyzed with InfoQuest FP software version 4.5 (Bio-Rad Laboratories Inc., Hercules, CA, USA). In order to establish the epidemiological relationship among the isolates from the electrophoretic patterns, the Dice coefficient was used and clustering was based on the unweighted pair group method with arithmetic mean with a 1% tolerance in band position differences. The isolates were considered to belong to the same epidemiological group when the PFGE-XbaI profiles showed ≥80% of homology, adapting the criteria described by Tenover et al.35 (link) Multi-locus sequence typing (MLST) was carried out by amplification and sequencing of the 7 E. coli housekeeping genes as described previously.36 (link) The database available at http://mlst.warwick.ac.uk/mlst/dbs/Ecoli/ was used for assigning sequence types (STs) and clonal complexes (CCs). Classification of isolates into E. coli phylogenetic groups was done using a previously described triplex PCR-based protocol.37 (link)
10
Pulsed-Field Gel Electrophoresis for Salmonella
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PFGE was performed on DS1, DS2, and RS to determine if the isolates were from the challenge strains or any extraneous Salmonella introduced during the study. At each timepoint, we randomly selected two isolates (from two different chickens) per group for analysis. PFGE was performed according to CDC’s PulseNet protocol.18 (link) DNA was digested with 50 U of XbaI restriction enzyme (New England Biolabs, Ipswich, MA, USA) for at least 2 h at 37°C. Salmonella Braenderup H9812 was used as a molecular reference marker. Electrophoresis was performed using CHEF-DR® III Pulsed-Field Electrophoresis System (Bio-Rad Laboratories, Hercules, CA, USA) with the following conditions and reagents: 1% SeaKem Gold agarose (FMC BioProducts, Rockland, Maine, USA) in 0.5% Tris-borate EDTA buffer, temperature: 14°C; voltage: 6 V/cm; run time: 18 h with switch times ranging from 2.2 to 63.8 s. The gels were stained with ethidium bromide and the DNA bands were visualized under UV trans-illumination (Gel Doc™ 2000, Bio-Rad Laboratories, Hercules, CA, USA) and gel images were captured using the Quantity one 1-D analysis software (Bio-Rad Laboratories, Hercules, CA, USA). PFGE gels were consolidated and visualized using Bionumerics software V. 4.61 (Applied Maths NV, Belgium).
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