The pCMV-PE2 was purchased from Addgene (132775) [15 (link)]. The PE2 plasmid was linearized by the PmeI enzyme (NEB, Ipswich, MA, USA), and in vitro transcription was performed using the T7 Ultra Kit (Ambion, Austin, TX, USA). According to the manufacturer’s protocols, RNA was purified by the Mini Kit (Qiagen, Hilden, Germany). The expression plasmids of T7 + pegRNA and T7 + nick sgRNA were linearized by the XhoI enzyme (NEB, Ipswich, MA, USA). According to the manufacturer’s protocols, the pegRNAs and sgRNAs were transcribed in vitro by the MEGA shortscript Kit (Invitrogen, Carlsbad, CA, USA) and purified by the MEGA clear Kit (Invitrogen, Carlsbad, CA, USA).
Xhoi enzyme
Manufactured by New England Biolabs
Sourced in United States
XhoI is a type II restriction enzyme that recognizes and cleaves the DNA sequence 5'-CTCGAG-3'. It is commonly used in molecular biology applications such as DNA cloning and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Recommended buffer, by New England Biolabs (2 mentions)
Megashortscript kit, by Thermo Fisher Scientific (1 mentions)
T7 ultra kit, by Thermo Fisher Scientific (1 mentions)
Pmei enzyme, by New England Biolabs (1 mentions)
Megaclear kit, by Thermo Fisher Scientific (1 mentions)
Pcmv pe2, by Addgene (1 mentions)
Plasmid preparation kit, by Beyotime (1 mentions)
Noti enzyme, by New England Biolabs (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Psicheck 2 vector, by Promega (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
5 protocols using xhoi enzyme
1
Plasmid Linearization and In Vitro Transcription
2
Quantifying Restriction Enzyme Cleavage
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These constructs were made as described above, but with a bridge oligonucleotide containing an inserted sequence recognized by the XhoI enzyme (New England Biolabs). The compliment to this restriction sequence was also added to ensure that this region was double stranded. Cleavage measurements were performed by adding enzyme to the loops (with final concentrations of 2.2 nM and 1,000 units/mL for the loops and enzyme, respectively) in the recommended buffer (New England Biolabs) and quenching the enzyme activity with 75 mM EDTA at various times at room temperature.
3
Dnmt3b Knockdown Plasmid Construction
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The mice Dnmt3b mRNA sequence was obtained from GenBank. The sequence was 5′-GCATGAAGGCCAGATCAAA-3′, which was designed and synthesized by GenePharma (Shanghai, China). The scrambled sequence was 5′-UUCUCCGAACGUGUCACGU-3′. BamHI and EcoRI enzymes (NEB) were used to digest and linerize the vector pLVX-shRNA1. The structure of the vector was demonstrated in Figure S3A (supplemental files). The products were mixed with competent cells JM109 which were further cultured on LB medium containing Ampicillin. A Plasmid Preparation kit (Beyotime) was used to extract the recombinant plasmids which were further identified by XhoI enzyme (NEB) digestion and DNA sequencing.
4
Characterizing PLD1 3'UTR-miR-122 Interaction
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A PLD1 3′UTR fragment was inserted into a psiCHECK-2 vector (Promega, USA) to build a psiCHECK-2-PLD1 3′UTR-wt construct. The psiCHECK-2-PLD1 3′UTR vector was nicked with Xho I enzyme (New England Biolabs, USA) and Not I enzyme (New England Biolabs). Q5 Site-Directed Mutagenesis Kit (New England Biolabs) was used to mutate in site 1761 psiCHECK-2-PLD1 3′UTR-wt to produce psiCHECK-2-PLD1 3′UTR-mut. NRK-52E and HK-2 cells were plated at a density of 1 × 105 cells per well in a 96-well plate and allowed to attach for 24 h. The constructed dual-luciferase plasmid psiCHECK-2-PLD1 3′UTR-wt, psiCHECK-2-PLD1 3′UTR-mut, or the control luciferase plasmid (100 ng/well) was cotransfected with miR-122-5p mimics or NC mimics for 36 h using Lipofectamine 3000 reagent (Life Technologies, USA). Luciferase activity was assayed 36 h after transfection using a Dual-Luciferase Reporter Assay Kit (Promega, USA). Experiments were repeated five times.
5
Quantifying Restriction Enzyme Cleavage
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