Poly(ethylene glycol) monomethyl ether MW 5000 kDa (mPEG), medium molecular weight chitosan (190–340 kDa), phthalic anhydride, anhydrous pyridine, 4-dimethylamino-pyridine (DMAP), succinic anhydride, anhydrous N,N-dimethylformamide (DMF), hydrazine monohydrate, hydroxybenzotrizole (HOBt), mucin from porcine stomach type III, egg yolk from chicken, and lactoferrin human were purchased from Sigma-Aldrich (St. Louis, MO, USA). Low molecular weight chitosan (oligosaccharide) (LMW OCs), 15 kDa, was obtained from Polysciences Inc., USA. Dulbecco’s Modified Eagle’s Medium (DMEM), Opti-MEM, and diethyl ether were obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Carbodiimide hydrochloride (EDC·HCl) and deuterium chloride were obtained from ACROS Organics (Morris Plains, NJ, USA). Deuterium oxide (D2O) and dimethyl sulfoxide-d6 were obtained from EMD Millipore Corporation (Billerica, MA, USA). pSpCas9-2A-GFP plasmid was purchased from Addgene (Plasmid #48138).
Pspcas9 2a gfp plasmid
Manufactured by Addgene
Sourced in United States
The PSpCas9-2A-GFP plasmid is a tool for gene editing. It expresses Cas9 protein and GFP marker from a single promoter. This plasmid can be used for CRISPR-Cas9 genome engineering experiments.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide d6, by Merck Group (1 mentions)
Deuterium oxide d2o, by Merck Group (1 mentions)
Anhydrous pyridine, by Merck Group (1 mentions)
Human lactoferrin, by Merck Group (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Diethyl ether, by Thermo Fisher Scientific (1 mentions)
Phthalic anhydride, by Merck Group (1 mentions)
Deuterium chloride, by Thermo Fisher Scientific (1 mentions)
Gel extraction kit, by Qiagen (1 mentions)
2 protocols using pspcas9 2a gfp plasmid
1
Chitosan-PEG Conjugate Synthesis
2
CRISPR-mediated Gene Editing of TRAC and CD52
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The gene sequences of human TRAC and CD52 genes were downloaded from NCBI and ensemble websites and gRNAs were designed to target the first exon of each gene using the CRISPR Design tool (http://crispr.mit.edu ). For each gRNA, two complementary 5′-phosphorylated oligonucleotides encompassing the gRNA sequence and BbsI restriction endonuclease site overhangs were synthesized (Table 1 ), annealed and sub-cloned into pSpCas9-2A-GFP plasmid (pX458, Addgene 48,138#, Cambridge, MA, USA) that had been digested with BbsI (New England Biolabs, Beverly, MA, USA), using a Golden Gate assembly cloning strategy [45 (link)], and gel purified using a Gel Extraction Kit (Qiagen). The resultant constructs were subjected to Sanger sequencing to verify proper sub-cloning of the gRNA sequences.
Oligos used for introduction of gRNA sequences for TRAC and CD52 genes into pSpCas9-2A-GFP plasmid
| Name | Sequence |
|---|---|
TRAC-gRNA (F/top) TRAC-gRNA (R/bottom) | 5′p-CACCG 5′p-AAACGCCGTGTACCAGCTGAGAGAC-3′ |
CD52-gRNA (F/top) CD52-gRNA (R/bottom) | 5′p-CACCG 5′p-AAACGTACCATAACCAGGAGGCTGC-3′ |
The gRNA sequences are underlined
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