sgRNAs were designed using the CRISPRtool (https://zlab.bio/guide-design-resources ). shRNA sequences were designed using SPLASH algorithm (http://splashrna.mskcc.org/ ; Pelossof et al, 2017 ) or the RNAi Consortium/Broad Institute (http://www.broadinstitute.org/rnai-consortium/rnai-consortium-shrna-library ). For site‐directed mutagenesis, guidance and instructions from the kit GeneEditor™ in vitro Site‐Directed Mutagenesis System (Promega) were followed.
Geneeditor in vitro site directed mutagenesis system
Manufactured by Promega
Sourced in United States
The GeneEditor in vitro Site-Directed Mutagenesis System is a tool designed for introducing specific modifications or mutations into DNA sequences. It provides a straightforward and efficient method for generating targeted genetic changes in plasmid or PCR-amplified DNA.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Pbluescript, by Promega (1 mentions)
Gateway pentr vector, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
5 protocols using geneeditor in vitro site directed mutagenesis system
1
CRISPR-Cas9 and shRNA Design Guidelines
2
Modulating CagA Expression in H. pylori Infection
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The GFP-MAP1LC3B plasmid and RFP-MAP1LC3B expression plasmid were kindly provided by Dr. Tamotsu Yoshimori (Department of Cell Biology, National Institute for Basic Biology, Presto, Japan) and Dr. Maria Colombo (Universidad Nacional de Cuyo, Mendoza, Argentina), respectively. The CagA expression plasmid, pEGFP-C1-CagA (GFP-CagA) (Asahi et al., 2000 (link); Suzuki et al., 2009 (link)), was kindly provided by Dr. Chihiro Sasakawa. The cagA mutant plasmid, pEGFP-C1-CagA-Mut (GFP-CagA-Mut) was constructed by Life Technologies, Shanghai, China, and a series of CagA mutants with the Tyr residues of 899, 918, and 972 being substituted by Ala were generated from a plasmid-encoding fragment of cagA gene of H. pylori ATCC 26695 on pBluescript (Promega, Madison, USA) using a Gene Editor in vitro Site-Directed Mutagenesis System (Promega). These mutants were at the sites 2,695–2,697, 2,752–2,754, and 2,914–2,915 bp, respectively, started from the sequence ATG. Lipofectamine 2000 (Invitrogen, #11668019) was used to transfect plasmids and/or siRNAs into AGS cells. 3 × 106 AGS cells were seeded into a 100-mm dish and incubated with transfection complexes containing 100 nM siRNA for 24 h.
3
In Vitro Site-Directed Mutagenesis of SMYD3
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Point mutants were generated using GeneEditor in vitro Site-Directed Mutagenesis System (Promega) according to manufacturer's instructions using as template full length human SMYD3 cloned into Gateway pENTR vector (Invitrogen). For PCR, samples were heated to 94°C for 5 min, subjected to amplification for 16 cycles of 0.5 min at 94°C, 0.5 min at 55°C, and 0.5 min at 68°C and extended after the last cycle at 72°C for 7 min.
4
Dual-Luciferase Assay for miR-498-5p Targets
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To confirm the putative miR-498-5p binding sites in HOXB-AS3 or ADAM9, relative luciferase activity was measured using the dual-luciferase reporter assay system (Promega, Madison, WI, USA). pGL4 luciferase reporter vectors were constructed for wild type (WT) HOXB-AS3 fragments (HOXB-AS3 WT) with miR-498-5p binding sites and HOXB-AS3 mutant (HOXB-AS3 MUT) without miR-498-5p binding sites. Mutants were generated using a GeneEditor in vitro Site-Directed Mutagenesis System (Promega). Ishikawa cells were co-transfected with HOXB-AS3 WT or HOXB-AS3 MUT and NC miRNA mimic or miR-498-5p mimic using Lipofectamine 2000 Transfection Reagent (Invitrogen). Similarly, WT or mutant ADAM9 3ʹ untranslated region (3ʹ UTR) with or without miR-498-5p binding sites were also cloned into pGL4 luciferase reporter vectors to form reporter vector ADAM9 WT and ADAM9 MUT, respectively. The relative luciferase activity was measured according to the manufacturer’s suggested protocol.
5
Site-directed mutagenesis of promoter plasmids
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The full-length promoter plasmids of LEF, FN, COX2, and COL1A1 were used as templates for site-directed mutagenesis using a GeneEditor in vitro site-directed mutagenesis system (Promega, Madison, WI, USA) to obtain various mutant promoters according to the manufacturer’s protocol. The bases changed in the site-directed mutagenesis for the proposed Snail binding motif (TCACA), and the EGR1/SP1 overlapping binding region were the same as those described in our previous report [17 (link),18 (link)].
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