H9 human embryonic stem cells were cultured on Vitronectin Recombinant Human Protein (Life Technologies) in Essential 8 medium (E8, Life Technologies) as described previously16 (link). Cells were passaged every three days as clumps with 0.5 mM EDTA (Lonza). For transfection, 2×106 cells were nucleofected using AmaxaTM P3 Primary Cell 4D-Nucleofector (Lonza) as recommended by the manufacturer, with no more than a 10 uL mix of 2 ug of plasmid carrying each gRNA, 2 ug of plasmid carrying hCas9 and 2–4 ug of plasmid carrying the donor DNA to repair the Cas9/gRNA induced break by homologous recombination. Cells were plated and allowed to recover for a minimum of 6h in E8 media supplemented with 2 uM thiazovivin (Stemgent). Drug selection with 1 ug/mL puromycin (InvivoGen) started 48h after transfection and lasted 2 days for loss of function cell lines, or continued for several days for gain of function cell line. Colonies were picked 10 days after selection and genotyped by PCR to confirm homozygosity. To generate the point mutations in the p63BSPM line, gRNAs were synthesized via in vitro transcription and transfected into cells with Lipofectamine CRISPRMAX (Invitrogen) along with Cas9 protein. The donor sequence supplied alongside was designed as a 200 bp single-stranded DNA oligo (an ssODN ultramer from IDT)43 ,44 .
Amaxa p3 primary cell 4d nucleofector
Manufactured by Lonza
The Amaxa™ P3 Primary Cell 4D-Nucleofector is a laboratory instrument designed for the transfection of primary cells. It utilizes Nucleofection technology to efficiently deliver nucleic acids, such as plasmids, RNA, or other molecules, into difficult-to-transfect cell types. The core function of this product is to facilitate the introduction of genetic material into primary cells for various research applications.
Automatically generated - may contain errors
2 protocols using amaxa p3 primary cell 4d nucleofector
1
CRISPR-Mediated Genetic Modifications in H9 hESCs
2
CRISPR-Mediated Genetic Modifications in H9 hESCs
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H9 human embryonic stem cells were cultured on Vitronectin Recombinant Human Protein (Life Technologies) in Essential 8 medium (E8, Life Technologies) as described previously16 (link). Cells were passaged every three days as clumps with 0.5 mM EDTA (Lonza). For transfection, 2×106 cells were nucleofected using AmaxaTM P3 Primary Cell 4D-Nucleofector (Lonza) as recommended by the manufacturer, with no more than a 10 uL mix of 2 ug of plasmid carrying each gRNA, 2 ug of plasmid carrying hCas9 and 2–4 ug of plasmid carrying the donor DNA to repair the Cas9/gRNA induced break by homologous recombination. Cells were plated and allowed to recover for a minimum of 6h in E8 media supplemented with 2 uM thiazovivin (Stemgent). Drug selection with 1 ug/mL puromycin (InvivoGen) started 48h after transfection and lasted 2 days for loss of function cell lines, or continued for several days for gain of function cell line. Colonies were picked 10 days after selection and genotyped by PCR to confirm homozygosity. To generate the point mutations in the p63BSPM line, gRNAs were synthesized via in vitro transcription and transfected into cells with Lipofectamine CRISPRMAX (Invitrogen) along with Cas9 protein. The donor sequence supplied alongside was designed as a 200 bp single-stranded DNA oligo (an ssODN ultramer from IDT)43 ,44 .
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