Abi1 cDNA was collected from pCR2.1 T/A cloning plasmid encoding human Abi1 and subcloned to pLenti-puro (Addgene #39481) at EcoRI and XhoI sites. Plasmids (pLenti-puro in BL21DE3plysS, pVSVG in DH5α, and pCMV in Stble3) were purified using the HiPure Plasmid Maxiprep Kit (Invitrogen). To produce viruses, 293FT cells were transfected with pLenti-puro encoding Plk1 plus packaging vector pCMV and envelop vector pVSV-G. Viruses were collected 48 h after transfection. For infection, smooth muscle cells were incubated with viruses 12 h, then cultured in the F12 growth medium for 2 days. Positive clones were selected by puromycin. Protein expression was assessed by immunoblotting.
Hipure plasmid maxiprep kit
Manufactured by Thermo Fisher Scientific
The HiPure Plasmid Maxiprep kit is a tool designed for the purification of high-quality plasmid DNA from bacterial cultures. It utilizes a column-based method to isolate and concentrate plasmid DNA, suitable for downstream applications such as transfection, sequencing, and cloning.
Automatically generated - may contain errors
Lab products found in correlation
Zymopure plasmid dna isolation kit, by Zymo Research (4 mentions)
Subcloning efficiency dh5α competent cells, by Thermo Fisher Scientific (3 mentions)
Long cdna synthesis, by Thermo Fisher Scientific (3 mentions)
Plenti puro, by Addgene (1 mentions)
Geneticin g418, by Thermo Fisher Scientific (1 mentions)
Sequabrene, by Merck Group (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Genejuice, by Merck Group (1 mentions)
Plko 1 puro empty vector shc001, by Merck Group (1 mentions)
Dsred2, by Takara Bio (1 mentions)
In fusion hd cloning kit, by Takara Bio (1 mentions)
Plko 1 vector, by Merck Group (1 mentions)
Quikchange site directed mutagenesis kit, by Thermo Fisher Scientific (1 mentions)
Quikchange site directed mutagenesis kit, by Agilent Technologies (1 mentions)
8 protocols using hipure plasmid maxiprep kit
1
Overexpression of Human Abi1 in SMCs
2
Overexpressing Human PDE5A1 in HEK293 Cells
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HEK293 cells were grown in DMEM supplemented with 10% FBS, in 75 mm flasks at 37 °C in a humidified 5%CO2. A human PDE5A1 plasmid, a gift from Professor Dr Joseph A. Beavo, University of Washington, Seattle, WA, USA, were sub‐cloned into a pcDNA3 vector containing an ampicillin resistant gene. The human PDE5-A1 plasmid was scaled up and purified using Hipure plasmid Maxiprep kit (Invitrogen‐PureLink). HEK293 cells were transfected with human PDE5A1 plasmid using Lipofectamine-2000 following the company protocol. After 2 days of transfection, PDE5-A1 expression was induced by a selective antibiotic (Geneticin (G418, Gibco), 1 mg/ml) for 7 days. The surviving cells were sub‐cultured in DMEM, supplemented with 10% FBS in 175 mm flasks at 37 °C in a humidified 5% CO2 atmosphere, and the cells further cultured until they reached 90–100% confluence. The cells were then harvested using a scraper and lysed by sonication in 1 ml of Tris buffer [50 mM Tris pH 7.5, 2 mM EDTA, 1mM dithiothreitol (DTT) and 1:100 of 100 mM PMSF]. The homogenate was centrifuged at 4 °C for 20 min and the supernatant was used as a source of PDE5A1. A PDE5 inhibitor, sildenafil, was used to confirm the presence of PDE5A1 enzymatic activity.
3
Generation and Transduction of SIM2 Constructs
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Cell lines were generated as previously described [13 (link)]. In brief, SIM2 constructs were generated via long cDNA synthesis. Plasmids were amplified using Subcloning Efficiency™ DH5α™ competent cells (Life Technologies). Plasmid DNA was isolated using the HiPure Plasmid Maxiprep kit (Life Technologies) or the ZymoPURE Plasmid DNA Isolation Kit (Zymo Research). Ten micrograms of plasmid was mixed with GeneJuice (EMD Millipore) in 1 mL of Opti-MEM (Life Technologies) and incubated at room temperature for 15 min. This mixture was then added onto Phoenix-AMPHO lentiviral packaging cells (ATCC). Cells were incubated for 24 h at 32 °C and 5% CO2. Media was collected and filtered through a 0.45-μm filter. The recommended amount of Sequabrene (Sigma) was added to the filtered media. The media was then added to SUM159 cells in six-well plates. Plates were centrifuged at 200×g for 60 min and allowed to incubate overnight at 32 °C and 5% CO2. Media was again collected from the packaging cells the next day, and target cells were transduced a second time, as described above. Puromycin selection (2 μg/mL) was started the following day and maintained for at least a week [14 (link)].
4
Generating Stable SIM2 Mutant Cell Lines
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Point mutations in the SIM2 gene were generated via long cDNA synthesis (Invitrogen). Plasmids were amplified using Subcloning Efficiency™ DH5α™ competent cells (Life Technologies). Plasmid DNA was isolated using the HiPure Plasmid Maxiprep kit (Life Technologies) or the ZymoPURE Plasmid DNA Isolation Kit (Zymo Research). Viral transduction was then preformed as previously described39 (link). Puromycin selection (2μg/mL) was started the following day and maintained for a week.
5
Mutagenesis of SIM2 Gene via cDNA Synthesis
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Point mutations in the SIM2 gene were generated via long cDNA synthesis (Invitrogen). Plasmids were amplified using Subcloning Efficiency DH5a competent cells (Life Technologies). Plasmid DNA was isolated using the HiPure Plasmid Maxiprep Kit (Life Technologies) or the ZymoPURE Plasmid DNA Isolation Kit (Zymo Research). Viral transduction was performed as previously described [38 (link)]. Puromycin selection (2 μg/mL) was started the following day and maintained for a week.
6
Generating Stable SIM2 Mutant Cell Lines
7
Lentiviral Vectors for TTL Expression
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For lentiviral experiments, vector eGFP-pWPT (Addgene no. 12255, kind gift from D. Trono) was used to express eGFP, and cDNA encoding human TTL (NP_714923, Origene no. RC207805L2) was cloned in it for TTL expression. PCR amplification and cloning of TTL cDNA were performed with Phusion DNA polymerase (Thermo Scientific) and In-Fusion HD Cloning kit (Clontech), respectively. eGFP cDNA was removed during the cloning process to produce an untagged TTL. For lentiviral shRNA expression, two TTL shRNA sequences, cloned in pLKO.1 vector, were purchased from Sigma-Aldrich: shTTL1 (TRCN0000191515, sequence: 5′-CCG GCA TTC AGA AA GAG TAC TCA ACT CGA GTT GAC TAC TCT TTC TGA ATG CTT TTT TG-3′) and shTTL2 (TRCN0000191227, sequence: 5′-CCG GCT CAA AGA ACT ATG GGA AAT ACT CGA GTA TTT CCC ATA GTT CTT TGA GTT TTT TG-3′).35 The SHC001 pLKO.1-puro empty Vector (Sigma) was used as control (shControl). For the transfection experiments, the plasmid encoding pCMV-EB3-EGFP was a kind gift from Dr Frank Polleux.72 (link) Kind gifts from Dr Erik Dent include the plasmids EB3-tdTomato (Addgene no. 50708) and the plasmid encoding DsRed2 (Clontech), cloned into a pCAX vector. The plasmid pEGFP-N1 with a CMV promoter was also used (Addgene no. 6085-1). All constructs were verified by sequencing (Eurofins and Genewiz). Plasmids were purified with HiPure Plasmid Maxiprep kits (Invitrogen).
8
Site-Directed Mutagenesis of mDia1 and Tau
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mDia1 shRNA 1 resistant (R), I845A, and K994A point mutants were generated on pmCherry-C1-mDia1 using the QuikChange Site-Directed Mutagenesis kit (Agilent Technologies) according to the manufacturer’s protocol and using the following synthetic sense oligonucleotide primers: 5′-CCTGCGACGGGCGGCGATGGAGGAAAACATAAGAAATTTACT-3′ (R), 5′-GACAGCGCAGAATCTCTCAGCCTTTTTGGGTTCATTCCGC-3′ (I845A), and 5′-TTGTAAGCTTCGAGACACCGCGTCTGCAGATCAGAAGATG-3′ (K994A). Human 2N4R S262A mutant tau was generated on human EGFP-tau2N4R tau using the QuikChange Site-Directed Mutagenesis kit according to the manufacturer’s protocol and using the synthetic sense oligonucleotide primer 5′-TTCAGGTTCTCAGTGGCGCCGATCTTGGACTTG-3′. All constructs were verified by sequencing, and the cDNAs were purified with HiPure Plasmid Maxiprep kits (Invitrogen).
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