Human GeCKOv2 CRISPR knockout pooled library was a gift from Feng Zhang (Addgene #1000000048) and human Brunello CRISPR knockout pooled library was a gift from David Root and John Doench (Addgene #73179). Library plasmids (1 µl) were electroporated into 20 µl MegaX DH10B T1 electrocompetent cells (Thermo Fisher Scientific) using a GenePulser II (BioRad; Settings: 2.0 kV, 200 Ω and 25 µF) in four replicates. Cells were resuspended in 2 ml S.O.C. recovery medium (Thermo Fisher Scientific) and incubated for 1 h at 37 °C and 250 rpm. Replicates were pooled and plated on two 245 mm × 245 mm plates (Corning) with ampicillin selection (100 µg/ml), which yielded 200 × (GeCKO sublibrary A) 800× (GeCKO sublibrary B) and 500× (Brunello) library coverage. After 14 h of incubation at 37 °C, colonies were scraped off and combined, and plasmid DNA was extracted using Nucleobond Xtra Maxi EF (Macherey Nagel).
Megax dh10b t1 electrocompetent cells
Manufactured by Thermo Fisher Scientific
Sourced in United States
MegaX DH10B T1 electrocompetent cells are a strain of genetically engineered Escherichia coli bacteria that have been made competent for efficient electroporation-based transformation. They are designed for high-efficiency DNA transformation in molecular biology applications.
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Lab products found in correlation
2 protocols using megax dh10b t1 electrocompetent cells
1
CRISPR Knockout Library Generation
2
Cloning and Expression of CreiLOV Variants
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E. coli DH5α cells were used for cloning and propagation of pET28
expression plasmids harboring WT CreiLOV or site-saturation mutants. E. coli MegaX DH10B T1 electrocompetent cells
(Thermo Fisher Scientific, MA, USA) were used for combinatorial library
construction. E. coli BL21(DE3)
cells were used for the expression of CreiLOV variants for fluorescence
screening. Cells were cultured in Luria broth (LB) medium (10 g/L
tryptone, 5 g/L yeast extract, 5 g/L NaCl, pH 7.0) with the supplement
of 50 μg mL–1 kanamycin at 37 °C and
220 rpm or on 1.5% LB agar plates and incubated at 37 °C.
expression plasmids harboring WT CreiLOV or site-saturation mutants. E. coli MegaX DH10B T1 electrocompetent cells
(Thermo Fisher Scientific, MA, USA) were used for combinatorial library
construction. E. coli BL21(DE3)
cells were used for the expression of CreiLOV variants for fluorescence
screening. Cells were cultured in Luria broth (LB) medium (10 g/L
tryptone, 5 g/L yeast extract, 5 g/L NaCl, pH 7.0) with the supplement
of 50 μg mL–1 kanamycin at 37 °C and
220 rpm or on 1.5% LB agar plates and incubated at 37 °C.
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