Human hERG K+ channel cloned into SP64 vector (kindly provided by Dr. Eduardo Perozo—University of Chicago). Mutations were performed using Quick-change II technology (Stratagene, La Jolla, CA), together with custom primers from Integrated DNA Technologies (Integrated DNA Technologies, Inc., Coralville, IA). hERG cDNA and its mutants were sequenced to ensure accurate DNA sequencing, linearized by endonuclease EcoRI (New England Biolabs, Ipswich, MA) and cleaned up with a NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Bethlehem, PA). In vitro transcription kits were used to transcribe cDNA and generate cRNA (SP6 RNA expression kit; Ambion Invitrogen, Thermo Fisher Scientific, Waltham, MA).
Ecori endonuclease
Manufactured by New England Biolabs
Sourced in United States
EcoRI endonuclease is a type II restriction enzyme that recognizes and cleaves the DNA sequence 5'-GAATTC-3'. It is a widely used tool in molecular biology for the restriction digestion of DNA.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Nucleospin gel and pcr clean up kit, by Macherey-Nagel (1 mentions)
Monarch pcr and dna cleanup kit, by New England Biolabs (1 mentions)
Hybond xl membrane, by GE Healthcare (1 mentions)
Minelute virus vacuum kit, by Qiagen (1 mentions)
T5 exonuclease, by New England Biolabs (1 mentions)
α 32p utp, by PerkinElmer (1 mentions)
T4 ligase, by New England Biolabs (1 mentions)
5975c mass selective detector, by Agilent Technologies (1 mentions)
Taq pcr reagent kit, by Thermo Fisher Scientific (1 mentions)
Vector nti advance 9, by Thermo Fisher Scientific (1 mentions)
Dneasy plant mini kit, by Qiagen (1 mentions)
Universal dna purification kit, by Tiangen Biotech (1 mentions)
Gel extraction kit, by Omega Bio-Tek (1 mentions)
7 protocols using ecori endonuclease
1
hERG Channel Cloning and Mutagenesis
2
Linearization and Purification of pUC19 Plasmid
3
Comprehensive HBV Nucleic Acid Extraction
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Total cellular RNA, cytoplasmic encapsidated HBV pgRNA, core DNA, and protein-free Hirt DNA were extracted as described previously (38 (link), 39 (link)). Non-cccDNA in Hirt DNA was removed via T5 exonuclease (New England BioLabs) digestion. EcoRI endonuclease (New England BioLabs) was then used to linearize HBV cccDNA before electrophoresis.
For HBV RNA Northern blot analysis, total cellular RNA or encapsidated pgRNA samples were resolved in a 1.5% agarose gel containing 2.2 M formaldehyde and transferred onto a Hybond-XL membrane (GE Healthcare). For DNA Southern blot analysis, HBV core DNA or cccDNA samples were resolved by electrophoresis in a 1.2% agarose gel and blotted onto a Hybond-XL membrane. Membranes were probed with either an [α-32P]UTP (3,000 Ci/mmol; PerkinElmer)-labeled plus-strand-specific (for Northern blot hybridization) or minus-strand-specific (for Southern blot hybridization) HBV riboprobe and exposed to a phosphorimager screen. Extracellular HBV DNA and RNA were copurified from the culture medium of induced HepAD38 cells by using a MinElute virus vacuum kit (Qiagen). Viral DNA was quantified using the above-mentioned qPCR method directly, and HBV RNA was detected by RT-qPCR with the same core region primer/probe set after the removal of DNA contamination by DNase I (Thermo Fisher) treatment.
For HBV RNA Northern blot analysis, total cellular RNA or encapsidated pgRNA samples were resolved in a 1.5% agarose gel containing 2.2 M formaldehyde and transferred onto a Hybond-XL membrane (GE Healthcare). For DNA Southern blot analysis, HBV core DNA or cccDNA samples were resolved by electrophoresis in a 1.2% agarose gel and blotted onto a Hybond-XL membrane. Membranes were probed with either an [α-32P]UTP (3,000 Ci/mmol; PerkinElmer)-labeled plus-strand-specific (for Northern blot hybridization) or minus-strand-specific (for Southern blot hybridization) HBV riboprobe and exposed to a phosphorimager screen. Extracellular HBV DNA and RNA were copurified from the culture medium of induced HepAD38 cells by using a MinElute virus vacuum kit (Qiagen). Viral DNA was quantified using the above-mentioned qPCR method directly, and HBV RNA was detected by RT-qPCR with the same core region primer/probe set after the removal of DNA contamination by DNase I (Thermo Fisher) treatment.
4
Contextual Analysis of bla_PER-1 Gene
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In order to investigate the immediate genetic context of the blaPER-1 gene, inverse-PCR and sequencing were performed with some modifications (Opazo et al., 2012 (link)). Briefly, the whole DNA was restricted with EcoRI endonuclease for 2 h at 37°C and then circularized using the T4 ligase for 16 h at room temperature following the manufacturer’s recommendations (New England Biolabs, Hertfordshire, UK). To determine the downstream context of blaPER-1 the circularized DNA was used as a template for a PCR using the primers invPER-F (5′-GCCGAACCAATGAAGCTATCATTGCGCAGG-3′) and invPER-R (5′-AATTTGCTCTTTTAACAGTGGGGATTGCGCTG-3′). The PCR products obtained were sequenced and analyzed. The upstream region of blaPER-1 gene was characterized as before (Opazo et al., 2012 (link)).
5
Quantifying DNA Base Modifications
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Calf thymus DNA was dissolved in buffer containing 5 mM NaCl, 1 mM Tris (pH 7), 1 mM MgCl2, and 0.1 mM DDT. DNA (∼50 mg) was digested with ∼20,000 units of EcoRI endonuclease (New England Biolabs) at 37 °C for 4 h to reduce viscosity (64 (link)). Digested DNA was precipitated with ammonium acetate/ethanol, resuspended in buffer, and dialyzed overnight.
A portion of the digested calf thymus DNA was hydrolyzed in 88% formic acid at 140 °C for 40 min. Isotope-enriched standards of thymine (T + 4), cytosine (C + 2), and 5-mC (5-mC + 3) at a ratio of 20:1 (C/5-mC) were added to the vials, which were then evaporated to dryness under reduced pressure. Bases were converted to the TBDMS derivatives in acetonitrile at 140 °C for 40 min. Samples were injected onto an Agilent 7890A GC containing a DB5 column. The initial GC oven temperature was 100 °C for 2 min, ramped to 260 °C at 30 °C per min, and then held at 260 °C for 10 min. The GC was directly interfaced to an Agilent 5975C mass selective detector, and data were collected in the selected ion mode. Molar amounts of C and T were determined by comparing experimental peak areas to standard curves. The molar amount of 5-mC was determined by comparing peak areas of unenriched C and 5-mC to peak areas of the isotope-enriched standards. Base composition determinations were done in triplicate.
A portion of the digested calf thymus DNA was hydrolyzed in 88% formic acid at 140 °C for 40 min. Isotope-enriched standards of thymine (T + 4), cytosine (C + 2), and 5-mC (5-mC + 3) at a ratio of 20:1 (C/5-mC) were added to the vials, which were then evaporated to dryness under reduced pressure. Bases were converted to the TBDMS derivatives in acetonitrile at 140 °C for 40 min. Samples were injected onto an Agilent 7890A GC containing a DB5 column. The initial GC oven temperature was 100 °C for 2 min, ramped to 260 °C at 30 °C per min, and then held at 260 °C for 10 min. The GC was directly interfaced to an Agilent 5975C mass selective detector, and data were collected in the selected ion mode. Molar amounts of C and T were determined by comparing experimental peak areas to standard curves. The molar amount of 5-mC was determined by comparing peak areas of unenriched C and 5-mC to peak areas of the isotope-enriched standards. Base composition determinations were done in triplicate.
6
Molecular Characterization of C. sinensis
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Laboratory common reagents such as ethanol, sucrose, paraformaldehyde, hematoxylin, eosin, agarose and electrophoresis reagents, etc. were purchased from Beijing Bioland Technology Company. Mercuric chloride (0.1%) for surface sterilization of freshly collected C. sinensis specimens was a gift from the Institute of Microbiology, Chinese Academy of Sciences. EcoRI endonuclease is a product of New England BioLabs, United States. The Universal DNA Purification kit was a product of TIANGEN BIOTECH Company, China. The DNeasy Plant Mini Kit was a product of Qiagen Company, Germany. The Gel Extraction Kit was a product of Omega Bio-Tek, United States. The Taq PCR reagent kit and Vector NTI Advance 9 software were purchased from Invitrogen, United States. Calmodulin was a product of Abicom (Shanghai, China).
7
Genomic DNA Extraction and Southern Blot
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Embryos were then placed in 1.5 mL Eppendorf tubes (200–300 per tube) in 500 µL HOM buffer (80 mM EDTA, 100 mM Tris-HCl, 0.5% SDS, pH 8.0) with 10 µL Proteinase K (0.5 mg/mL) and incubated at 55°C for 3 h, vibrating once per hour. Genomic DNA was then extracted with phenol/chloroform. Roche DIG-High Primer DNA Labelling and Detection Starter Kit I (Hoffman-La Roche Ltd., Basel, Switzerland; catalog number 11745832910) was used for DNA fragment detection. Genomic DNA was digested overnight at 37°C with 4 units of EcoRI endonuclease per microgram of DNA, according to manufacturer’s instructions (New England Biolabs, Ipswich, MA, USA; R310S), and then electrophoresed on a 1% agarose gel prepared with Tris-borate-EDTA (TBE). Then, the DNA was stained with ethidium bromide and visualized by UV illumination. Next, the DNA was denatured using NaOH and transferred to nylon membranes (Roche; LOT15569600). The digoxigenin (DIG)-labeled probe was prepared according to the kit instructions. Normal zebrafish embryonic DNA was used as a negative control, and the SV40 sequence was used as a positive control.
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