The cDNA template was generated by isolation of mRNA from HepaRG™ progenitor cells (QIAGEN RNeasy Mini Kit) and subsequent reverse transcription (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems, Waltham, USA). cDNA was amplified by PCR (Thermo Scientific Phusion Green Hot Start II High-Fidelity Polymerase, Thermo Fisher, Waltham, USA) (CYP3A4-FW: 5′-ATATATGGTACCGCCACCATGGCTCTCATCCCA-3′, CYP3A4-RV: 5′-ATCTCGAGGGCTCCACTTACGGTGCCA-3′). The obtained PCR product was cloned into the pcDNA3-EGFP vector using FastDigest KpnI, FastDigest XhoI and T4 DNA Ligase (all purchased from Thermo Fisher) as indicated by the manufacturer. pcDNA3-EGFP was a gift from Doug Golenbock (Addgene plasmid #13031; http://n2t.net/addgene:13031 ; RRID:Addgene_13031). Correct insertion of the insert was confirmed by PCR, restriction digestion and Sanger sequencing. Sequencing services were provided by Eurofins Genomics (Munich, Germany). Primers were purchased from Metabion (Planegg, Germany).
Thermo scientific phusion green hot start 2 high fidelity polymerase
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Thermo Scientific Phusion Green Hot Start II High-Fidelity Polymerase is a DNA polymerase designed for high-fidelity amplification of DNA. It provides improved specificity and sensitivity compared to standard Taq DNA polymerases.
Automatically generated - may contain errors
Lab products found in correlation
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Fastdigest xhoi, by Thermo Fisher Scientific (1 mentions)
Fastdigest kpni, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Plasmid maxiprep kit, by Qiagen (1 mentions)
Pspcas9 bb 2a gfp px458 plasmid, by Addgene (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
2 protocols using thermo scientific phusion green hot start 2 high fidelity polymerase
1
Cloning CYP3A4 into pcDNA3-EGFP Vector
2
Generating TRPML1 KO B16F10-luc Cell Line
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The TRPML1 KO cell line in murine B16F10-luc cells was conducted with the CRISPR-Cas9 system as described earlier (86 (link)). To do so, we deleted exon 2 of the MCOLN1 gene. Then, single-guide RNAs used by Siow et al (2023) were cloned into the pSpCas9(BB)-2A-GFP (PX458) plasmid (Addgene, #48138). The plasmids were transformed into competent DH5α-Escherichia coli and subsequently prepared using the QIAGEN Plasmid Maxiprep Kit according to the manufacturer’s instructions. We then confirmed correct insertions by sequencing from the U6 promotor. After successful confirmation, B16F10-luc WT cells were transfected with both plasmids according to the Lipofectamine 3000 (Invitrogen) manufacturer’s instructions followed by single-cell sorting (Cell Sorter BD FACSAria Fusion) into 96-well plates and subsequent clonal expansion. Successful exon 2 deletion was confirmed by standard PCR (Thermo Scientific Phusion Green Hot Start II High-Fidelity Polymerase, Thermo Fisher Scientific), agarose gel analysis, and Sanger sequencing.
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