E coli xl10 gold cells

Manufactured by Agilent Technologies

Sourced in United Kingdom

E. coli XL10-Gold cells are a strain of Escherichia coli bacteria commonly used in molecular biology laboratories. They are designed to provide high transformation efficiency for various DNA manipulation and cloning applications.

Automatically generated - may contain errors

Lab products found in correlation

Miniprep kit, by Qiagen (1 mentions) Flag peptide, by Merck Group (1 mentions) Anti flag m2 monoclonal antibody, by Merck Group (1 mentions) Site directed mutagenesis kit, by Agilent Technologies (1 mentions) Yeast nitrogen base, by Merck Group (1 mentions) Bl21 codonplus de3 ril e coli cells, by Agilent Technologies (1 mentions) Pmaxgfp, by Lonza (1 mentions) Pcdfduet 1, by Merck Group (1 mentions) E coli bl21 de3, by Agilent Technologies (1 mentions) Dh5α cells, by New England Biolabs (1 mentions) Pet30a, by Merck Group (1 mentions) Pet21a, by Merck Group (1 mentions) Purexpress, by New England Biolabs (1 mentions) Dreamtaq dna polymerase mastermix, by Thermo Fisher Scientific (1 mentions) Egg pc, by Avanti Polar Lipids (1 mentions) Pcdna3, by Thermo Fisher Scientific (1 mentions) T4 ligase, by Promega (1 mentions)

Close spelling variants of this product

XL10-Gold (25 citations) XL10-Gold cells (16 citations) E. coli XL10-Gold (12 citations) XL10-Gold ultra-competent E. coli cells (3 citations)

7 protocols using e coli xl10 gold cells

Purification of Epitope-Tagged Human Topoisomerase I

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Anti-FLAG M2 monoclonal affinity gel, FLAG peptide, anti-FLAG M2 monoclonal antibody were purchased from Sigma-Aldrich and the antibody against the C-terminus of hTop1 from Abcam.
Saccharomyces cerevisiae Top1 null strain EKY3 (ura3-52, his3Δ200, leu2Δ1, trp1Δ63, top1::TRP1, MATα) was used to express the hTop1 gene. YCpGAL1-e-hTop1 single copy plasmid was previously described [20 (link)].
The 8bmut and the 8hmut mutants were generated using a site-directed-mutagenesis kit (Agilent Technologies) of the YCpGAL1-hTop1 in which the hTop1 is expressed under the galactose inducible promoter in a single-copy plasmid. The epitope-tagged construct YCpGAL1-e-hTop1 contains the N-terminal sequence FLAG: DYKDDDDY (indicated with ‘e’), recognized by the M2 monoclonal antibody. The epitope-tag was subcloned into YCpGAL1-hTop18bmut or YCpGAL1-hTop18hmut to produce the YCpGAL1-e-hTop18bmut and YCpGAL1-e-hTop18hmut. The plasmids were transformed into XL10-Gold E. coli cells (Agilent Technologies) and, then, extracted using Quiagen miniprep kit. Positive clones were identified by sequencing the hTop1 gene of the extracted plasmids. After the transformation in EKY3 yeast strain, the purification of hTop1 proteins was carried out essentially as previously described [21 (link)].

Tesauro C., Graziani G., Arnò B., Zuccaro L., Muzi A., D’Annessa I., Santori E., Tentori L., Leonetti C., Fiorani P, & Desideri A. (2014). Mutations of human DNA topoisomerase I at poly(ADP-ribose) binding sites: modulation of camptothecin activity by ADP-ribose polymers. Journal of Experimental & Clinical Cancer Research : CR, 33(1), 71.

+ Open protocol

+ Expand

Bacterial and Yeast Cell Culture Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

XL10-Gold E. coli cells (Agilent) and E. coli BL21-CodonPlus (DE3)-RIL cells (Agilent) were cultivated in luria broth (LB) or terrific broth (TB) medium (RPI). The Bg12 strain of P. pastoris (Biogrammatics) was cultured in buffered minimal glycerol (BMGY) or buffered minimal methanol (BMMY) media containing yeast nitrogen base (Sigma-Aldrich). All cell lines used in this study were authenticated by the suppliers and were chosen to remain consistent with previous studies.

Ferré G., Anazia K., Silva L.O., Thakur N., Ray A.P, & Eddy M.T. (2022). Global insights into the fine tuning of human A2AAR conformational dynamics in a ternary complex with an engineered G protein viewed by NMR. Cell reports, 41(12), 111844.

+ Open protocol

+ Expand

Plasmid Constructs for Insulin Receptor Studies

Check if the same lab product or an alternative is used in the 5 most similar protocols

Plasmids were maintained in E. coli XL10-Gold cells (Agilent Technologies, Stockport, UK; cat. no. 200314). Standard protocols for plasmid constructions were used (Ausubel et al., 2017 ). Plasmid pmaxGFP was obtained from Lonza Cologne AG (Cologne, Germany). Plasmid pEGFP-N2-hINSR encodes a fusion of the human insulin receptor to eGFP (Bass et al., 2000 (link)) and was obtained from Addgene (Cambridge, MA; Addgene ID 22286). Plasmid pcDNA5/FRT/TO-F_V2E-INSRβ was generated by cloning the 1430-base-pair BsiWI-XmaI fragment of pCLFv2IRE (Cotugno et al., 2004 (link)) into BsiWI- and XmaI-digested pcDNA5/FRT/TO-F_V2E-C’hIRE1α (D. Cox and M. Schröder, Durham University, Durham, UK, unpublished data). Plasmid pcDNA5/FRT/TO-MyrF_V2E-INSRβ was generated by cloning the 501-base-pair EcoRI-XmaI fragment of pC₄M-F_V2E (Ariad Pharmaceuticals, Cambridge, MA) into HindIII- and XmaI-digested pcDNA5/FRT/TO-F_V2E-INSRβ after blunting the EcoRI and HindIII sites with Klenow enzyme.

Brown M., Dainty S., Strudwick N., Mihai A.D., Watson J.N., Dendooven R., Paton A.W., Paton J.C, & Schröder M. (2020). Endoplasmic reticulum stress causes insulin resistance by inhibiting delivery of newly synthesized insulin receptors to the cell surface. Molecular Biology of the Cell, 31(23), 2597-2629.

+ Open protocol

+ Expand

Genetic Engineering Techniques in E. coli

Check if the same lab product or an alternative is used in the 5 most similar protocols

Escherichia coli B40 (sup⁺), BL21 (DE3) (Tyr sup⁺), BL21 (DE3) (Gly sup⁺), and BL21 (DE3) (Arg sup⁺) were used as amber suppressors for initial crosses and preparation of phage stocks. The sup^-E. coli P301 (B strain) and sup^-E. coli D21 (K12 strain) (E. coli Genomic Resource Center, Yale University, New Haven, CT) were used in marker rescue assays. E. coli BL21 (sup^-) was used as the host for pET28b plasmid carrying g37 alanine substitution mutants and mutant libraries. The E. coli XL10-Gold cells (Agilent Technologies, Santa Clara, CA) or DH5α cells (New England BioLabs, Ipswich, MA), were used for initial transformation and maintenance of recombinant constructs. For protein expression, sequence-confirmed recombinant clones were transferred into the expression strain E. coli BL21 (DE3) (Agilent Technologies, Santa Clara, CA) or E. coli BL21 (DE3) pLysS (Stratagene, La Jolla, CA) to allow IPTG (isopropyl-β-D-thiogalactopyranoside)-induced overexpression of recombinant proteins [53 (link)]. E. coli BZB1109 (a gift from Prof. Tillman Schirmer, Biozentrum, University of Basel, Basel, Switzerland) was used to express OmpC. The T7 expression plasmids pET28b, pCDFDuet-1, pET21a, and pET30a (EMD Millipore, MA), were used as the cloning vectors. Purified phage T4 genomic DNA was used as a template to amplify T4 g37 and its mutants.

Islam M.Z., Fokine A., Mahalingam M., Zhang Z., Garcia-Doval C., van Raaij M.J., Rossmann M.G, & Rao V.B. (2019). Molecular anatomy of the receptor binding module of a bacteriophage long tail fiber. PLoS Pathogens, 15(12), e1008193.

+ Open protocol

+ Expand

Recombinant Transketolase Purification and Characterization

Check if the same lab product or an alternative is used in the 5 most similar protocols

Wild-type transketolase with an N-terminal His6-tag was expressed in E. coli XL10-gold cells (Agilent Technologies Ltd) from the plasmid pQR791. The resulting cell pellet was lysed and purified as described previously^{37 (link)}. Purified transketolase was ultrafiltrated four times using Amicon Ultra-4 10k MWCO centrifugal filter to remove excess imidazole and cofactors and subsequently dialysed overnight at 4 °C in 50 mM Tris-HCl, pH 7.0 to obtain apo-TK. Protein concentration was determined by absorbance at 280 nm in 6 M Guanidine-HCl and 20 mM Sodium Phosphate, pH 6.5. Absorbance was measured using a Nanodrop spectrophotometer, assuming a monomeric molecular weight of 73035.5 g mol⁻¹ and an extinction coefficient of 92630 L mol⁻¹ cm⁻¹.
Series of 2x concentrated cofactor solutions were prepared and purified TK was added to a final concentration of either 0.05 mg/mL or 0.2 mg/mL. The samples were incubated at 22 °C for 45 minutes to allow TK-TPP binding to reach equilibrium.

Wilkinson H.C, & Dalby P.A. (2019). Novel insights into transketolase activation by cofactor binding identifies two native species subpopulations. Scientific Reports, 9, 16116.

+ Open protocol

+ Expand

Protein Modification and Characterization

Check if the same lab product or an alternative is used in the 5 most similar protocols

All consumables, solvents and reagents were purchased from Sigma/Merck unless stated otherwise. NHS–PCB–Biotin was purchased from Ambergen and stored in dry dimethylformamide at −80 °C. DreamTaq DNA polymerase master mix, TR-dextran (10 kDa) and 25 µl gene frames were purchased from Thermo Fisher Scientific. PURExpress and all other enzymes were purchased from New England Biolabs. E. coli XL10-Gold cells were purchased from Agilent. Egg PC was purchased from Avanti Polar Lipids. The 1 mm diamond drill bit was purchased from Eternal Tools. The M365L2-C5 UV lamp was purchased from Thorlabs. Standard DNA oligonucleotides were synthesized by integrated DNA technologies. Photomasks were designed using AutoCAD and purchased from JD Photodata. Amine-modified DNA oligonucleotides were synthesized by ATD-Bio. pMAT–mNG was synthesized by GeneArt. pSB1A3–bjaR–gfp was a gift from K. Haynes (Arizona State University). pET24a was a gift from B. Davis (University of Oxford). Monovalent streptavidin was a gift from M. Howarth (University of Oxford). All DNA sequences can be found in Supplementary Data.

Smith J.M., Hartmann D, & Booth M.J. (2023). Engineering cellular communication between light-activated synthetic cells and bacteria. Nature Chemical Biology, 19(9), 1138-1146.

+ Open protocol

+ Expand

Construction of Optogenetic Vectors

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cassettes were introduced in pcDNA3.1-(Invitrogen/Life Technologies) to generate the vectors named pOVC1-3 (optogenetic vector core 1-3, Figure S4). A XmaI restriction site was removed from the backbone using site-directed mutagenesis (oligonucleotides 1 and 2, Table S2). Inverse polymerase chain reactions (PCR) (oligonucleotides 3 and 4, 5 and 6, and 7 and 8) were applied to remove the vector multiple cloning site and create ABC (pOVC1), ACB (pOVC2) and BAC (pOVC3) cassettes. In the inverse PCR procedure, PCR products were digested with DpnI, digested with EcoRI, XmaI or AgeI (NEB), respectively, ligated for 3 h at room temperature (RT) or overnight at 4°C using T4 ligase (Promega), and propagated in E.coli XL10 Gold cells (Agilent). All cassettes contain Kozak sequences, start codons and stop codons (for backbone ABC, the stop codon was introduced using sitedirected mutagenesis in a separate reaction (oligonucleotides 9 and 10)). For linker insertion, backbone pOVC1 was digested using EcoRI and BamHI. Linker fragments were generated by inverse PCR (oligonucleotides 57 and 58) or by annealing and phosphorylating single stranded oligonucleotides (59 to 64). All vector sequences (Table S3) were verified by Sanger sequencing (Micromon, Monash University) and deposited at Addgene.org.

Tichy A.M., Gerrard E.J., Legrand J.M.D., Hobbs R.M, & Janovjak H. (2019). Engineering Strategy and Vector Library for the Rapid Generation of Modular Light-Controlled Protein-Protein Interactions. Journal of molecular biology, 431(17).

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!