DNA constructs were built using NEBuilder HiFi DNA Assembly Master Mix (New England BioLabs Cat. #E2621) and transformed into NEB 10-beta electrocompetent E.coli (New England BioLabs Cat. #3020). DNA was extracted using a Qiagen Plasmid Midi Kit (Qiagen Cat. #12143) and sequenced by Sanger sequencing at Genewiz. Primers used for cloning can be found in the Key resources table below.
Neb 10 beta electrocompetent e coli
Manufactured by New England Biolabs
NEB 10-beta electrocompetent E.coli is a laboratory strain of Escherichia coli bacteria that has been made electrocompetent, which means it has been treated to increase its ability to take up and incorporate foreign DNA through electroporation. This product is intended for use in molecular biology and genetic engineering applications where bacterial transformation is required.
Automatically generated - may contain errors
Lab products found in correlation
Plasmid midi kit, by Qiagen (4 mentions)
Nebuilder hifi dna assembly master mix, by New England Biolabs (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Pspax2, by Addgene (1 mentions)
Pmd2 g, by Addgene (1 mentions)
Gibson assembly master mix, by New England Biolabs (1 mentions)
Plasmid maxi kit, by Qiagen (1 mentions)
Gene pulser xcell electroporation system, by Bio-Rad (1 mentions)
7 protocols using neb 10 beta electrocompetent e coli
1
DNA Construct Assembly and Sequencing
2
Plasmid Construction and Transformation
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All plasmids were built following standard molecular biology approaches. Plasmids were constructed by Gibson Assembly using NEBuilder HiFi DNA Assembly Master Mix (New England BioLabs Cat. #E2621) and transformed into NEB 10-beta electrocompetent E.coli (New England BioLabs Cat. #3020). Plasmids were purified using a Qiagen Plasmid Midi Kit (Qiagen Cat. #12143), and DNA sequences were confirmed by Sanger sequencing. All constructions and their NCBI ID were shown in Supplementary Fig. 1 .
3
Lentiviral Transduction of CHO Cells
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Target plasmid was maintained in and purified from NEB 10-beta electrocompetent E. coli (New England Biolabs, C3020K). Lentivirus was packaged by plating 4×106 HEK293T cells on 10 cm2 and incubating cells overnight at 37°C. Cells were transfected with a plasmid mix consisting of 3.5 µg of the target plasmid, 6.0 µg psPAX2 (Addgene, 12260), and 3.0 µg pMD2.G (Addgene, 12259) using Lipofectamine 2000 (ThermoFisher Scientific, 11668019) in accordance with the manufacturer’s instructions. Transfected HEK293T cells were incubated for 48 hr, before medium was collected, and centrifuged at 200×g for 5 mins. The resulting supernatant was filtered using a 0.45 µm filter before infection. CHO cells were plated at 20–30% confluency in and incubated overnight prior to infection. The packaged virus was applied to cells for 24 hr before the medium was exchanged for fresh medium. After another 24 hr of incubation, the medium was exchanged for fresh medium containing 10 µg/ml puromycin for 8 days.
4
Plasmid construction using Gibson assembly
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All plasmids were cloned using standard molecular biology techniques. Plasmids were constructed by Gibson assembly using NEBuilder HiFi DNA Assembly Master Mix (New England BioLabs Cat. #E2621) and transformed into NEB 10-beta electrocompetent E.coli (New England BioLabs Cat. #3020). Plasmid DNA was prepared using a Qiagen Plasmid Midi Kit (Qiagen Cat. #12143) and sequences were confirmed by Sanger sequencing at Genewiz. Primers used for cloning can be found in Supplementary Information and the validated sequences of all constructs Have been deposited in the GenBank database; accession numbers are provided in the Supplementary Information and in the Data availability.
5
DNA Construct Assembly and Sequencing
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DNA constructs were built using NEBuilder HiFi DNA Assembly Master Mix (New England BioLabs Cat. #E2621) and transformed into NEB 10-beta electrocompetent E.coli (New England BioLabs Cat. #3020). DNA was extracted using a Qiagen Plasmid Midi Kit (Qiagen Cat. #12143) and sequenced by Sanger sequencing at Genewiz. Primers used for cloning can be found in the Key resources table below.
6
Barcoded Lentiviral Library Construction
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The construction of barcoded libraries was executed in accordance with a previously established protocol (https://www.protocols.io/view/barcode-plasmid-library-cloning-4hggt3w ). First, the T-Sapphire or EGFP coding sequences, and the EF1a promoter sequences were PCR amplified from pEB1-T-Sapphire and pLARRY-EGFP with primers homologous to the vector insertion site in a custom lentiviral plasmid backbone (Vectorbuilder, Inc) using Gibson assembly (Gibson Assembly® Master Mix, NEB, Ref. E2611L). After magnetic-bead purification, ligated vectors were then transformed into NEB10-beta electroporation ultracompetent E.coli cells (NEB® 10-beta Electrocompetent E. coli, NEB, Ref.C3020K) and grown overnight on LB plates supplemented with 50 μg/mL Carbenicillin (Carbenicillin disodium salt, Thermo Scientific Chemicals Ref. 11568616). Colonies were scrapped using LB medium and pelleted by centrifugation. Plasmid maxipreps were performed using the Endotoxin-Free Plasmid Maxi Kit (Macheray Nagel), following the manufacturer protocol. pEB1-T-Sapphire was a gift from Philippe Cluzel (Addgene plasmid 103977). pLARRY-EGFP was a gift from Fernando Camargo (Addgene plasmid 140025)
7
Membrane Protein Activation Library Protocol
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The human membrane protein activation library developed by G. Wright was obtained from Addgene (cat. no. 113345). The library DNA was electroporated into NEB 10-beta electrocompetent E. coli [New England Biolabs (NEB), cat. no. C3020K] at 2.0 kV, 200 Omega and 25 μF using a Gene Pulser Xcell Electroporation System (Bio-Rad). Transformed bacteria were then grown in 500 ml of 2× Yeast Extract Tryptone (YT) medium/amp (ampicillin; 50 μg/ml) medium and incubated at 37°C overnight with shaking at 220g. A Qiagen plasmid maxi kit (Qiagen, cat. no. 12163) was used to purify library DNA from 500 ml of bacteria culture following the manufacturer’s instruction. To determine the library representation after purification, 10 ng of purified plasmid DNA was used as template for polymerase chain reaction (PCR) amplification and sequencing using the Illumina MiSeq.
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