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13 protocols using stbl2 cells

1

Purification and Characterization of Telomeric DNA

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pSXneo(T2AG3) plasmid DNA containing 270 TTAGGG repeats was purchased from Addgene and was purified from Stbl2 cells (Invitrogen)52 (link). To generate linear DNA fragments containing TTAGGG repeats (T270) for AFM imaging, digestion of pSXneo(T2AG3) plasmid DNA (10 μg) was carried out at 37 °C for 4 hr using HpaI (130 U, NEB). The final digested product was purified using the QIAquick PCR Purification Kit (Qiagen). The electrophoresis mobility shift assays (EMSAs) were carried out as reported previously using an Alexa488 labeled 48-bp duplex DNA susbrate containing 3 TTAGGG repeats (Top strand: 5′[Alexa488]TTAGGGTTAGGGTTAGGGATGTCCAGCAAGCCAGAATTCGGCAGCGTA-3′)50 (link). The DNA substrate containing a mismatch was prepared as described previously51 (link)53 (link).
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2

Cloning and SECRETS Assays

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All cloning was performed using New England Biolabs (NEB) 10-beta cells (NEB #C3020K) or TOP10 (Invitrogen #C404010) cells, and all SECRETS assays performed in Stbl2 cells (Invitrogen #10268019), grown at 30°C. A549 (ATCC CCL-185) human lung epithelial cells were obtained from were obtained from ATCC (American Type Culture Collection).
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3

Heterologous Expression of Human SCN3A

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Transfections and electrophysiological experiments were performed using tsA-201 cells. Cells were grown at 37°C with 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and penicillin (50 U/mL)-streptomycin (50 μg/mL).
A plasmid encoding the major splice isoform of human SCN3A (isoform 2, which is 49 amino acids longer than isoform 1) was used (Reference Sequence NP_001075145.1), and variants were introduced by site-directed mutagenesis. Constructs were propagated in STBL2 cells at 30 °C (Invitrogen). All plasmid preparations were resequenced prior to transfection. Plasmids encoding human sodium channel auxiliary subunits β1 (hβ1-V5-2A-dsRed) or β2 (pGFP-IRES- hβ2) were co-transfected in vectors containing marker genes facilitating the identification of cells expressing all three constructs (Nav1.3, Navβ1, and Navβ2). We performed transient transfection of 2 μg total cDNA at a ratio of Nav1.3, &beta1, and β2 of 10:1:1 using Lipofectamine-2000 (Invitrogen) transfection reagent. Cells were incubated for 48 hours after transfection prior to electrophysiological recording. Transfected cells were dissociated by brief exposure to trypsin/EDTA, re-suspended in supplemented DMEM medium, plated on 15 mm glass coverslips, and allowed to recover for at least 4 hours at 37 °C in 5% CO2 prior to recording.
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4

Cloning and Assay Protocols

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All cloning was performed using New England Biolabs (NEB) 10-beta cells (NEB #C3020K) or TOP10 (Invitrogen #C404010) cells, and all SECRETS assays performed in Stbl2 cells (Invitrogen #10268019), grown at 30 °C. A549 (ATCC CCL-185™) human lung epithelial cells were obtained from ATCC (American Type Culture Collection).
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5

Mutational Analysis of WNV Structural Genes

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We used a previously described expression vector encoding the structural genes (C-prM-E) of the WNV NY99 strain15 (link) as a template for site-directed mutagenesis using the Pfu Ultra DNA polymerase system (Agilent Technologies). PCR cycling parameters were: 1 cycle of 95°C for 1 m; 18 cycles of 95°C for 50 s, 60°C for 50 s, and 68°C for 9 m; and 1 cycle of 68°C for 7 m. Following digestion with DpnI (New England Biolabs) for 3 h at 37°C, PCR products were used to transform Stbl2 cells (Invitrogen) and propagated at 30°C. After confirming the presence of the desired mutation by sequencing, the entire C-prM-E region was sequenced to ensure that no additional mutations were present.
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6

Heterologous Expression of Murine HCN1 Channels

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Methods for heterologous expression were as previously described [22 (link)]. Briefly, cDNA encoding murine HCN1
channels were sub-cloned into a pGH19 vector and amplified in STBL2 cells
(Invitrogen Corporation, Grand Island, NY, USA). cRNA was transcribed from NheI
linearized DNA using T7 RNA polymerase (Message Machine; Ambion, Houston, TX,
USA).
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7

Site-directed mutagenesis of flavivirus E proteins

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We used a previously described expression vector encoding the structural genes (C-prM-E) of the WNV NY99 strain [57 (link)] as a template for mutagenesis. Initially, threonine at residue 198 of the WNV E protein was replaced with phenylalanine by site-directed mutagenesis using the Pfu Ultra DNA polymerase system (Agilent Technologies). The reciprocal mutation (F193T) was introduced into a previously described expression vector encoding the structural genes (C-prM-E) of the DENV1 Western Pacific strain [86 (link)]. Mutation at the analogous residue of the ZIKV E protein (F198T) was introduced into a plasmid encoding the structural genes of the ZIKV strain H/PF/2013 [120 ]. This plasmid is described elsewhere [121 ]. Additional amino acid variants were introduced at position 198 of the WNV E protein using primers containing a degenerate codon (NNN). PCR cycling parameters were as follows: 1 cycle of 95°C for 1 min; 18 cycles of 95°C for 50 s, 60°C for 50 s, and 68°C for 9 min; and 1 cycle of 68°C for 7 min. PCR products were treated with DpnI (New England BioLabs) for 3 h at 37°C, prior to transformation into Stbl2 cells (Invitrogen) and propagation at 30°C. The entire C-prM-E region of each construct was sequenced to ensure that no additional mutations were present.
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8

HIV Pseudovirus Production and Quantification

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HEK293T (HEK, human embryonic kidney cells) were purchased from ATCC (Manassas, VA). The following reagents were obtained from the NIH AIDS Reference and Reagent Program, Division of AIDS, NIAID: HOS CD4+ CXCR4+ Cells from Dr. Nathaniel Landau; pDOLHIVenv from Dr. Eric O. Freed and Dr. Rex Risser; TZM-bl cells from Dr. John C. Kappes, Dr. Xiayun Wu and Tranzyme Inc.; CEM-GFP cells from Dr. Jacques Corbeil; pNL4-3 from Dr. Malcom Martin; and monoclonal antibody 16H3 (ARP-12559) from Duke Human Vaccine Institute. The pNL4-3 Luc AM pseudotyping backbone plasmid was a generous gift from Dr. John Moore. Dr. Joseph Sodroski donated the expression plasmid for HIV-1 HxBc2 strain Envelope. Escherichia coli strains XL-10 gold (Agilent) and Stbl2 cells (Invitrogen) were used for propagating DNA. Thermostable DNA polymerase (PfuUltra™) was obtained from Stratagene Inc. (La Jolla, CA). Phusion® High-Fidelity Polymerase was purchased from New England Biolabs (Ipswich, MA). Integrated DNA Technologies supplied Custom-oligonucleotide primers. Polyethylenimine (PEI) 25 kDa linear polymer, was obtained from Polysciences, Inc. (Warrington, PA). All other reagents were purchased from Sigma-Aldrich unless otherwise specified.
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9

Preparation and Use of HIV-1 Env Plasmids

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Escherichia coli strain XL-10 gold (Agilent) and Stbl2 cells (Invitrogen) were products of Novagen Inc. (Madison, WI). Thermostable DNA polymerase (PfuUltra) was obtained from Stratagene Inc. (La Jolla, CA). Custom-oligonucleotide primers were supplied by Integrated DNA Technologies (IDT). DNA plasmids encoding HIV-1BaL Env (catalog no. 11445) and NL4-3 R E Luc+ were obtained from the NIH AIDS Reagent Program, Division of AIDS, NIAID, and were a kind gift of Dr. J Mascola and Dr. N Landau, respectively. All other reagents used were of the highest analytical grade available.
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10

Mutational Analysis of WNV Structural Genes

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We used a previously described expression vector encoding the structural genes (C-prM-E) of the WNV NY99 strain15 (link) as a template for site-directed mutagenesis using the Pfu Ultra DNA polymerase system (Agilent Technologies). PCR cycling parameters were: 1 cycle of 95°C for 1 m; 18 cycles of 95°C for 50 s, 60°C for 50 s, and 68°C for 9 m; and 1 cycle of 68°C for 7 m. Following digestion with DpnI (New England Biolabs) for 3 h at 37°C, PCR products were used to transform Stbl2 cells (Invitrogen) and propagated at 30°C. After confirming the presence of the desired mutation by sequencing, the entire C-prM-E region was sequenced to ensure that no additional mutations were present.
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