cDNAs for soluble SARS-CoV-2 S and RBD (containing C-terminal His-tag) [11 (link)] were cloned into EcoRV-cut plenti-CMV-Puro-DEST (Addgene, #17452, gift from Eric Campeau & Paul Kaufman) using NEBuilder HiFi DNA Assembly Master Mix (NEB). Lentivirus was produced in HEK293FT by co-transfection of cDNA containing pLenti plasmids together with pCMV-dR8.2 dvpr (Addgene, #8455, gift from Bob Weinberg), pCMV-VSV-G (Addgene, #8454, gift from Bob Weinberg) and pAdVAntage (Promega) using FugeneHD (Promega). Supernatants were collected 48 hr post-transfection and filtered using a 0.45 μm syringe filter. For the transduction of Expi293 cells, five 6-well plates with 2 million cells per well were spin-infected with lentivirus diluted 1:2.5 in fresh Expi293 expression media under the following conditions: 2 hr, 33°C, 1,000 g. Cells were subsequently pooled together and cultured in 30 mL Expi293 expression media on a shaking incubator. 24 h post-transduction puromycin was added at 2 μg/mL and cells were selected for 7 days.
Ecorv cut plenti cmv puro dest
Manufactured by Addgene
The EcoRV-cut plenti-CMV-Puro-DEST is a plasmid vector designed for lentiviral gene delivery and expression. It contains the human cytomegalovirus (CMV) promoter for constitutive transgene expression and a puromycin resistance gene for selection of transduced cells.
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Lab products found in correlation
Nebuilder hifi dna assembly master mix, by New England Biolabs (3 mentions)
Pcmv vsv g, by Addgene (2 mentions)
Pcmv dr8.2 dvpr, by Addgene (2 mentions)
Padvantage, by Promega (2 mentions)
Fugene hd, by Promega (2 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Transit lenti, by Mirus Bio (1 mentions)
Pmd2 g, by Addgene (1 mentions)
Polybrene, by Merck Group (1 mentions)
Tmprss2, by Addgene (1 mentions)
Hace2, by Addgene (1 mentions)
Plenti dsred ires egfp, by Addgene (1 mentions)
Plenti cmv hygro dest, by Addgene (1 mentions)
Nebuilder hifi dna assembly master mix, by Addgene (1 mentions)
3 protocols using ecorv cut plenti cmv puro dest
1
Lentiviral Production and Transduction of Expi293 Cells
2
Cloning and Lentiviral Transduction of Human and Mouse CD164
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Human CD164 (number RC202234; Origene) and mouse Cd164 (number MR201951; Origene) cDNAs were cloned into EcoRV-cut plenti-CMV-Puro-DEST (Addgene number 17452; gift from Eric Campeau and Paul Kaufman) (61 (link)) using NEBuilder HiFi DNA assembly master mix (NEB). Primers used to assemble expression plasmids for domain deletion mapping and alanine scanning mutagenesis of CD164 can be found in Table S4 .
Lentivirus was produced in HEK293T cells by cotransfection of cDNA containing lentiviral plasmid together with helper plasmids pMD2.G (Addgene number 12259; gift from Didier Trono) and pCMV-dR8.91 (Life Science Market) using TransIT-Lenti (Mirus Bio). Supernatants were collected 48 h posttransfection, filtered, and added to recipient cells in the presence of Polybrene (EMD Millipore). Transduced cells were subsequently selected using puromycin (Thermo Fisher Scientific) during days 3 to 5.
Lentivirus was produced in HEK293T cells by cotransfection of cDNA containing lentiviral plasmid together with helper plasmids pMD2.G (Addgene number 12259; gift from Didier Trono) and pCMV-dR8.91 (Life Science Market) using TransIT-Lenti (Mirus Bio). Supernatants were collected 48 h posttransfection, filtered, and added to recipient cells in the presence of Polybrene (EMD Millipore). Transduced cells were subsequently selected using puromycin (Thermo Fisher Scientific) during days 3 to 5.
3
Lentivirus Production for SARS-CoV-2 Proteins
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The following cDNA sequence containing plasmids were obtained: hACE2 (Addgene, #1786, gift from Hyeryun Choe), TMPRSS2 (Addgene, #53887, gift from Roger Reeves), TMEM106B (Genscript, OHu17671) and VAC14 (Addgene, #47418, gift from Peter McPherson).
Individual cDNAs were cloned into EcoRV-cut plenti-CMV-Puro-DEST Addgene, #17452, gift from Eric Campeau & Paul Kaufman) or plenti-CMV-Hygro-DEST (Addgene, #17454, gift from Eric Campeau & Paul Kaufman) using NEBuilder HiFi DNA Assembly Master Mix (NEB). To generate the plenti-CMV-ACE2-IRES-TMPRSS2 construct, ACE2, EMCV IRES (derived from pLenti-DsRed_IRES_EGFP (Addgene, #92194, gift from Huda Zoghbi)), and TMPRSS2 were individually amplified with addition of overlapping sequences and the three fragments were assembled using NEBuilder HiFi DNA Assembly Master Mix.
Lentivirus was produced in HEK293FT by co-transfection of cDNA containing lentiviral plasmid together with pCMV-dR8.2 dvpr (Addgene, #8455, gift from Bob Weinberg), pCMV-VSV-G (Addgene, #8454, gift from Bob Weinberg) and pAdVAntage (Promega) using FugeneHD (Promega). Supernatants were collected 48h post-transfection, filtered and added to recipient cells in presence of Polybrene (SCBT). Transduced cells were subsequently selected using Puromycin or Hygromycin for 5–7 days.
Individual cDNAs were cloned into EcoRV-cut plenti-CMV-Puro-DEST Addgene, #17452, gift from Eric Campeau & Paul Kaufman) or plenti-CMV-Hygro-DEST (Addgene, #17454, gift from Eric Campeau & Paul Kaufman) using NEBuilder HiFi DNA Assembly Master Mix (NEB). To generate the plenti-CMV-ACE2-IRES-TMPRSS2 construct, ACE2, EMCV IRES (derived from pLenti-DsRed_IRES_EGFP (Addgene, #92194, gift from Huda Zoghbi)), and TMPRSS2 were individually amplified with addition of overlapping sequences and the three fragments were assembled using NEBuilder HiFi DNA Assembly Master Mix.
Lentivirus was produced in HEK293FT by co-transfection of cDNA containing lentiviral plasmid together with pCMV-dR8.2 dvpr (Addgene, #8455, gift from Bob Weinberg), pCMV-VSV-G (Addgene, #8454, gift from Bob Weinberg) and pAdVAntage (Promega) using FugeneHD (Promega). Supernatants were collected 48h post-transfection, filtered and added to recipient cells in presence of Polybrene (SCBT). Transduced cells were subsequently selected using Puromycin or Hygromycin for 5–7 days.
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