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).
Fastdigest kpni
Manufactured by Thermo Fisher Scientific
Sourced in United States
FastDigest KpnI is a restriction enzyme that recognizes and cleaves the DNA sequence 5'-GGTACC-3'. It is designed for fast and efficient DNA digestion, providing complete digestion in as little as 5 minutes. The enzyme is part of Thermo Fisher Scientific's FastDigest enzyme portfolio.
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)
Thermo scientific phusion green hot start 2 high fidelity polymerase, 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)
Geneart, by Thermo Fisher Scientific (1 mentions)
Pcdna3.4 topo vector, by Thermo Fisher Scientific (1 mentions)
Nanodrop one one, by Thermo Fisher Scientific (1 mentions)
One shot top10 chemically competent e coli, by Thermo Fisher Scientific (1 mentions)
Pvax1 vector, by Thermo Fisher Scientific (1 mentions)
Zymopure 2 plasmid maxiprep kit, by Zymo Research (1 mentions)
4 protocols using fastdigest kpni
1
Cloning CYP3A4 into pcDNA3-EGFP Vector
2
Fluorescent Protein Gene Cloning
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The genes encoding the fluorescent proteins mCherry (NCBI Accession AY678264.1) and mCerulean (NCBI Accession KP666136.1) were PCR amplified and cloned into the pFB1 cyto and SS-R vectors of the 1st generation cloning system. Moreover, the EGFP coding sequence was amplified from the pFastBac1 EGFP-µNS vector and ligated into the pFB1 v2 cyto, NLS, SS, SS-R, MTS1 and MTS2 vectors, after digestion with FastDigest KpnI and NheI (Thermo Scientific).
3
Engineered Telomere Enrichment Vectors
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The ReDACT-TR technique was motivated by the use of a telomere seed sequence to generate monosomic cells as described in ref1 (link). To enhance the efficacy of this approach, we created new plasmids linking a telomere seed sequence with a puromycin selection marker, which allowed us to enrich for stably-transfected aneuploidy-loss cells using drug selection. These EF1a-Puro-Telo vectors (Addgene #195138 and #195139) were generated by introducing a puromycin selection marker to a telomere seed sequence gifted by Alison Taylor. This vector was digested with FastDigest KpnI (Thermo Fisher Scientific, cat. no. FD0524) and FastDigest BstZ17I (Thermo Fisher Scientific, cat. no. FD0704), and gel purified to obtain the artificial telomere construct.
4
Plasmid DNA Purification and Quantification
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All DNA sequences were synthesized at GeneArt (ThermoFisher Scientific, Waltham, MA, USA) and cloned into the pcDNA3.4-TOPO vector (ThermoFisher Scientific, Waltham, MA, USA). In addition, they were subcloned into the pVAX1 vector (ThermoFisher Scientific, Waltham, MA, USA) using FastDigest KpnI and XhoI restriction enzymes (ThermoFisher Scientific, Waltham, MA, USA). All plasmids were transformed into One Shot TOP10 Chemically Competent E. coli (Invitrogen) for plasmid DNA amplification. Plasmids were purified in endotoxin-free conditions using the ZymoPure II Plasmid Maxiprep Kit (Zymo Research) and sterile filtered at 0.22 µm (Millipore). Nucleic acid concentration was measured using NanoDrop One/One (ThermoFisher Scientific, Waltham, MA, USA), based on the absorbance at 260 nm.
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