Structured Review

Thermo Fisher dh5α
Promoter activity in native and genetically engineered nlpD1 - penA intergenic regions. ( a ) Design of reporter lacZ reporter gene constructs. The indicated 238 bp DNA fragments shown by the thick lines (plus flanking SpeI and HindIII restriction sites) were obtained from PCR fragments amplified from 1026b and Bp1651 genomic DNA. The indicated −41C, −78G and −171T nucleotides present on the 1026b fragment were individually changed to −41T, −78A and −171C present in Bp1651. Lastly, a 532 bp DNA fragment (plus flanking SpeI and HindIII restriction sites) containing a 336 bp instead of a 42 bp nlpD1 terminus was obtained from a PCR fragment amplified from 1026b genomic DNA. These fragments were cloned into a mini-Tn 7 - lacZ transcriptional fusion vector and integrated into the Bp82.27 chromosome. ( b ) β-Gal activity in strains harboring the single-copy lacZ reporter constructs. The strains resulting from chromosomal integration were Bp82.363 (empty vector), Bp82.365 (1026b fragment), Bp82.365 (Bp1651 fragment), Bp82.366 (1026b fragment with −41T), Bp82.367 (1026b fragment with −78A), Bp82.371 (1026b fragment with −171C) and Bp82.372 (1026b fragment with a 331 bp instead of 42 bp nlpD1 3′ terminus. β-Gal activities were measured in triplicate on three separate days and are expressed in Miller units. Error bars indicate standard deviation from the mean. ( c ) The P A promoter is active in E . coli . A plasmid was constructed that contains a penA ’-‘ lacZ translational fusion under transcriptional and translation control of the 174 bp 1026b IR with the -78 G to A transition. The resulting pPZ10 penA ’-‘ lacZ thus contains the P A promoter. The first 7 amino acids of PenA are fused in-frame to LacZ. The resulting pPZ10 penA ’-‘ lacZ and the empty pPZ10 vector were transformed into strain <t>DH5α.</t> β-Gal activity was assessed by plating on an LB plate with X-Gal indicator (left) or by measuring hydrolysis of ortho-nitrophenyl-β-D-galactopyranoside (ONPG) in culture grown cells (right). The ONPG β-Gal assays were performed in triplicate and activity is expressed in Miller units. Error bars indicate standard deviation from the mean. The plate picture is a minimally processed section of an image of an agar plate shown in Fig. S6 .
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Images

1) Product Images from "Transcriptional and post-transcriptional regulation of PenA β-lactamase in acquired Burkholderia pseudomallei β-lactam resistance"

Article Title: Transcriptional and post-transcriptional regulation of PenA β-lactamase in acquired Burkholderia pseudomallei β-lactam resistance

Journal: Scientific Reports

doi: 10.1038/s41598-018-28843-7

Promoter activity in native and genetically engineered nlpD1 - penA intergenic regions. ( a ) Design of reporter lacZ reporter gene constructs. The indicated 238 bp DNA fragments shown by the thick lines (plus flanking SpeI and HindIII restriction sites) were obtained from PCR fragments amplified from 1026b and Bp1651 genomic DNA. The indicated −41C, −78G and −171T nucleotides present on the 1026b fragment were individually changed to −41T, −78A and −171C present in Bp1651. Lastly, a 532 bp DNA fragment (plus flanking SpeI and HindIII restriction sites) containing a 336 bp instead of a 42 bp nlpD1 terminus was obtained from a PCR fragment amplified from 1026b genomic DNA. These fragments were cloned into a mini-Tn 7 - lacZ transcriptional fusion vector and integrated into the Bp82.27 chromosome. ( b ) β-Gal activity in strains harboring the single-copy lacZ reporter constructs. The strains resulting from chromosomal integration were Bp82.363 (empty vector), Bp82.365 (1026b fragment), Bp82.365 (Bp1651 fragment), Bp82.366 (1026b fragment with −41T), Bp82.367 (1026b fragment with −78A), Bp82.371 (1026b fragment with −171C) and Bp82.372 (1026b fragment with a 331 bp instead of 42 bp nlpD1 3′ terminus. β-Gal activities were measured in triplicate on three separate days and are expressed in Miller units. Error bars indicate standard deviation from the mean. ( c ) The P A promoter is active in E . coli . A plasmid was constructed that contains a penA ’-‘ lacZ translational fusion under transcriptional and translation control of the 174 bp 1026b IR with the -78 G to A transition. The resulting pPZ10 penA ’-‘ lacZ thus contains the P A promoter. The first 7 amino acids of PenA are fused in-frame to LacZ. The resulting pPZ10 penA ’-‘ lacZ and the empty pPZ10 vector were transformed into strain DH5α. β-Gal activity was assessed by plating on an LB plate with X-Gal indicator (left) or by measuring hydrolysis of ortho-nitrophenyl-β-D-galactopyranoside (ONPG) in culture grown cells (right). The ONPG β-Gal assays were performed in triplicate and activity is expressed in Miller units. Error bars indicate standard deviation from the mean. The plate picture is a minimally processed section of an image of an agar plate shown in Fig. S6 .
Figure Legend Snippet: Promoter activity in native and genetically engineered nlpD1 - penA intergenic regions. ( a ) Design of reporter lacZ reporter gene constructs. The indicated 238 bp DNA fragments shown by the thick lines (plus flanking SpeI and HindIII restriction sites) were obtained from PCR fragments amplified from 1026b and Bp1651 genomic DNA. The indicated −41C, −78G and −171T nucleotides present on the 1026b fragment were individually changed to −41T, −78A and −171C present in Bp1651. Lastly, a 532 bp DNA fragment (plus flanking SpeI and HindIII restriction sites) containing a 336 bp instead of a 42 bp nlpD1 terminus was obtained from a PCR fragment amplified from 1026b genomic DNA. These fragments were cloned into a mini-Tn 7 - lacZ transcriptional fusion vector and integrated into the Bp82.27 chromosome. ( b ) β-Gal activity in strains harboring the single-copy lacZ reporter constructs. The strains resulting from chromosomal integration were Bp82.363 (empty vector), Bp82.365 (1026b fragment), Bp82.365 (Bp1651 fragment), Bp82.366 (1026b fragment with −41T), Bp82.367 (1026b fragment with −78A), Bp82.371 (1026b fragment with −171C) and Bp82.372 (1026b fragment with a 331 bp instead of 42 bp nlpD1 3′ terminus. β-Gal activities were measured in triplicate on three separate days and are expressed in Miller units. Error bars indicate standard deviation from the mean. ( c ) The P A promoter is active in E . coli . A plasmid was constructed that contains a penA ’-‘ lacZ translational fusion under transcriptional and translation control of the 174 bp 1026b IR with the -78 G to A transition. The resulting pPZ10 penA ’-‘ lacZ thus contains the P A promoter. The first 7 amino acids of PenA are fused in-frame to LacZ. The resulting pPZ10 penA ’-‘ lacZ and the empty pPZ10 vector were transformed into strain DH5α. β-Gal activity was assessed by plating on an LB plate with X-Gal indicator (left) or by measuring hydrolysis of ortho-nitrophenyl-β-D-galactopyranoside (ONPG) in culture grown cells (right). The ONPG β-Gal assays were performed in triplicate and activity is expressed in Miller units. Error bars indicate standard deviation from the mean. The plate picture is a minimally processed section of an image of an agar plate shown in Fig. S6 .

Techniques Used: Activity Assay, Construct, Polymerase Chain Reaction, Amplification, Clone Assay, Plasmid Preparation, Standard Deviation, Transformation Assay

2) Product Images from "KPC-4 Is Encoded within a Truncated Tn4401 in an IncL/M Plasmid, pNE1280, Isolated from Enterobacter cloacae and Serratia marcescens"

Article Title: KPC-4 Is Encoded within a Truncated Tn4401 in an IncL/M Plasmid, pNE1280, Isolated from Enterobacter cloacae and Serratia marcescens

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.01062-12

Plasmid analysis and corresponding bla KPC Southern blot. (A) Lane 1 (from left), 1-kb DNA ladder (Life Technologies, Grand Island, NY); lane 2, E. cloacae plasmid DNA digested with PstI; lane 3, E. coli DH5α 1623 plasmid DNA digested with PstI;
Figure Legend Snippet: Plasmid analysis and corresponding bla KPC Southern blot. (A) Lane 1 (from left), 1-kb DNA ladder (Life Technologies, Grand Island, NY); lane 2, E. cloacae plasmid DNA digested with PstI; lane 3, E. coli DH5α 1623 plasmid DNA digested with PstI;

Techniques Used: Plasmid Preparation, Southern Blot

3) Product Images from "Characterization of Hemolytic Escherichia coli Strains in Ferrets: Recognition of Candidate Virulence Factor CNF1"

Article Title: Characterization of Hemolytic Escherichia coli Strains in Ferrets: Recognition of Candidate Virulence Factor CNF1

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.42.12.5904-5908.2004

Ferret NTEC strains exhibit CNF1-mediated cytopathic effects. Subconfluent monolayers of HeLa cells were treated with sonicates of ferret NTEC strains. After 72 h, the cells exhibited distention and > 80% were multinucleated. Nuclear fragmentation was also apparent in some cells. Representative monolayers of HeLa cells treated with a sonicate of ferret NTEC strain 02-0042 (A) and a sonicate of the nonpathologic strain DH5α (B) and an untreated control monolayer (C) are shown at the same magnification after being stained with Diff-Quik.
Figure Legend Snippet: Ferret NTEC strains exhibit CNF1-mediated cytopathic effects. Subconfluent monolayers of HeLa cells were treated with sonicates of ferret NTEC strains. After 72 h, the cells exhibited distention and > 80% were multinucleated. Nuclear fragmentation was also apparent in some cells. Representative monolayers of HeLa cells treated with a sonicate of ferret NTEC strain 02-0042 (A) and a sonicate of the nonpathologic strain DH5α (B) and an untreated control monolayer (C) are shown at the same magnification after being stained with Diff-Quik.

Techniques Used: Staining, Diff-Quik

4) Product Images from "Treponema pallidum 3-Phosphoglycerate Mutase Is a Heat-Labile Enzyme That May Limit the Maximum Growth Temperature for the Spirochete"

Article Title: Treponema pallidum 3-Phosphoglycerate Mutase Is a Heat-Labile Enzyme That May Limit the Maximum Growth Temperature for the Spirochete

Journal: Journal of Bacteriology

doi: 10.1128/JB.183.16.4702-4708.2001

The effect of temperature on the complementation of E. coli strain PL225 ( gpm ). Strains DH5α, PL225, and PL225 harboring pSLB2 were grown overnight, harvested by centrifugation, washed twice in M63, and used to inoculate the fresh M63 medium. Gpm synthesis was induced from plasmid pSLB2 with 1 mM IPTG. Cultures were grown at 34 or 42°C, and cell density was monitored at 600 nm. Samples were run in duplicate, and data represent three independent experiments.
Figure Legend Snippet: The effect of temperature on the complementation of E. coli strain PL225 ( gpm ). Strains DH5α, PL225, and PL225 harboring pSLB2 were grown overnight, harvested by centrifugation, washed twice in M63, and used to inoculate the fresh M63 medium. Gpm synthesis was induced from plasmid pSLB2 with 1 mM IPTG. Cultures were grown at 34 or 42°C, and cell density was monitored at 600 nm. Samples were run in duplicate, and data represent three independent experiments.

Techniques Used: Centrifugation, Plasmid Preparation

5) Product Images from "Functional replacement of the endogenous tyrosyl-tRNA synthetase-tRNATyr pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion"

Article Title: Functional replacement of the endogenous tyrosyl-tRNA synthetase-tRNATyr pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq080

E. coli DH5α cells, an E. coli K-12 strain, expressing the wild-type E. coli TyrRS from plasmid pEcYS (filled circles) and those expressing iodoTyrRS- ec from plasmid pEcIYS (open circles) were grown in the presence of 3-bromo- l -tyrosine (0.5 mg/ml) ( A ) and 3-azido- l -tyrosine (0.3 mg/ml) ( B ). FT3 cells expressing iodoTyrRS- ec were grown in the absence (filled circles) and presence (open circles) of 3-bromo- l -tyrosine ( C ). The growth was monitored upon the addition of the non-natural amino acid to the growth media.
Figure Legend Snippet: E. coli DH5α cells, an E. coli K-12 strain, expressing the wild-type E. coli TyrRS from plasmid pEcYS (filled circles) and those expressing iodoTyrRS- ec from plasmid pEcIYS (open circles) were grown in the presence of 3-bromo- l -tyrosine (0.5 mg/ml) ( A ) and 3-azido- l -tyrosine (0.3 mg/ml) ( B ). FT3 cells expressing iodoTyrRS- ec were grown in the absence (filled circles) and presence (open circles) of 3-bromo- l -tyrosine ( C ). The growth was monitored upon the addition of the non-natural amino acid to the growth media.

Techniques Used: Expressing, Plasmid Preparation

Related Articles

Clone Assay:

Article Title: Aggregative Adherence and Intestinal Colonization by Enteroaggregative Escherichia coli Are Produced by Interactions among Multiple Surface Factors
Article Snippet: .. We used E. coli K-12 strains TOP10 and DH5α (Invitrogen) as host strains for cloned genes unless otherwise indicated. ..

Article Title: Deletion of ptn1, a PTEN/TEP1 Orthologue, in Ustilago maydis Reduces Pathogenicity and Teliospore Development
Article Snippet: .. Escherichia coli strains TOP10 and DH5α (Invitrogen/Thermo Fisher) were used for all cloning and plasmid maintenance. ..

Article Title: Transcriptional and post-transcriptional regulation of PenA β-lactamase in acquired Burkholderia pseudomallei β-lactam resistance
Article Snippet: .. Bacterial strains and growth Escherichia coli strains used in this study were DH5α (ThermoFisher Scientific, Waltham, MA) and XL1 Blue (Agilent Technologies, Santa Clara, CA) for routine cloning and RHO3 for conjugation . ..

Article Title: Transcriptional and post-transcriptional regulation of PenA β-lactamase in acquired Burkholderia pseudomallei β-lactam resistance
Article Snippet: .. Escherichia coli strains used in this study were DH5α (ThermoFisher Scientific, Waltham, MA) and XL1 Blue (Agilent Technologies, Santa Clara, CA) for routine cloning and RHO3 for conjugation . ..

Article Title: Analysis of the Behavior of eryC Mutants of Brucella suis Attenuated in Macrophages
Article Snippet: .. The Escherichia coli strains CC118λpir ( ) and DH5α (Invitrogen, Cergy Pontoise, France) were used as host strains for the cloning experiments with the erythritol gene of B. suis ; they were grown in Luria-Bertani broth in the presence of kanamycin or ampicillin at a concentration of 50 μg/ml when appropriate. .. Constructions were performed using plasmids pUC4K, pUC18 (both Amersham Biosciences, Orsay France), pGEM-T Easy (Promega, Charbonnières, France), pBBR1MCS , and pCVD442 ( ).

Centrifugation:

Article Title: A Novel, Molybdenum-Containing Methionine Sulfoxide Reductase Supports Survival of Haemophilus influenzae in an In vivo Model of Infection
Article Snippet: The resulting ligation products were transformed into DH5α (Life Technologies) to obtain pProex HItorZ -SP, which was verified by DNA sequencing. .. IPTG was added to a final concentration of 100 μM and the cultures incubated at 30°C, 200 rpm overnight before harvesting of cells by centrifugation (3000 × g , 4°C, 10 min).

Article Title: Treponema pallidum 3-Phosphoglycerate Mutase Is a Heat-Labile Enzyme That May Limit the Maximum Growth Temperature for the Spirochete
Article Snippet: E. coli strains used in this study were TOP10 (Invitrogen, Carlsbad, Calif.); DH5α (Gibco BRL, Grand Island, N.Y.), and PL225 ( gpm ): Δ( nadA-galE ) 35 λ− recA1 relA1 rpsL180 (Strr ) spoT1 thi-1 ( ). .. For complementation assays, strains DH5α, PL225, and PL225 harboring pSLB2 were grown overnight in LB medium at 30°C, harvested by centrifugation (5,000 × g , 10 min), and washed twice in M63, and 30 μl was used to inoculate 30 ml of M63 with 0.2% glucose.

Synthesized:

Article Title: Alteration in the cavity size adjacent to the active site of RB69 DNA polymerase changes its conformational dynamics
Article Snippet: Materials All oligonucleotides were synthesized at the Keck facilities (Yale University) and purified via polyacrylamide gel electrophoresis. .. Escherichia coli strain BL21 GOLD (DE3) and DH5α were from Invitrogen.

Construct:

Article Title: Specificity of the Type II Secretion Systems of Enterotoxigenic Escherichia coli and Vibrio cholerae for Heat-Labile Enterotoxin and Cholera Toxin ▿
Article Snippet: Plasmids were conjugated into V. cholerae by triparental mating as described previously , using DH5α (Invitrogen) containing the appropriate plasmid as the donor strain and MT616 carrying pRK2013 as the helper strain. .. Similar levels of expression of wild-type and mutant LT, LTB, or CTB constructs were detected after induction with a final concentration of 200 μM isopropyl-1-thio-β- d -galactopyranoside (IPTG; Sigma), except for the CTB[Q3K] construct, which required induction with 50 μM IPTG.

Article Title: A Novel, Molybdenum-Containing Methionine Sulfoxide Reductase Supports Survival of Haemophilus influenzae in an In vivo Model of Infection
Article Snippet: Construction of a TorZ Overexpression Plasmid A pProexHtb (Invitrogen) based protein expression plasmid containing the torZ gene without the region coding for the signal peptide was constructed using primers HIbisC-SP_pPro_Bam_F and HIbisC_pPro_Xba_R ( Table ) and the restriction enzyme sites introduced during high fidelity PCR. .. The resulting ligation products were transformed into DH5α (Life Technologies) to obtain pProex HItorZ -SP, which was verified by DNA sequencing.

Article Title: KPC-4 Is Encoded within a Truncated Tn4401 in an IncL/M Plasmid, pNE1280, Isolated from Enterobacter cloacae and Serratia marcescens
Article Snippet: Plasmid was isolated from E. cloacae and E. coli using either the method of Kado and Liu ( ) or the Qiagen (Valencia, CA) Large Construct Kit following the manufacturer's recommendations. .. Plasmid DNA was transformed into E. coli ElectroMAX Stbl4 or DH5α (Invitrogen, Carlsbad, CA) using heat shock and the manufacturer's recommendations.

Incubation:

Article Title: A Novel, Molybdenum-Containing Methionine Sulfoxide Reductase Supports Survival of Haemophilus influenzae in an In vivo Model of Infection
Article Snippet: The resulting ligation products were transformed into DH5α (Life Technologies) to obtain pProex HItorZ -SP, which was verified by DNA sequencing. .. Overexpression cultures (100 mL supplemented LB in a 250 mL shake flask) were inoculated from overnight cultures to an OD600 of 0.05–0.1, followed by incubation with shaking at 37°C, with shaking at 200 rpm until an OD600 of 0.6–0.8 was reached.

Article Title: KPC-4 Is Encoded within a Truncated Tn4401 in an IncL/M Plasmid, pNE1280, Isolated from Enterobacter cloacae and Serratia marcescens
Article Snippet: Plasmid DNA was transformed into E. coli ElectroMAX Stbl4 or DH5α (Invitrogen, Carlsbad, CA) using heat shock and the manufacturer's recommendations. .. Following 18 h of incubation at 37 ° C, the filter was placed in 0.5 ml saline, vortexed, diluted, and plated on LB agar (Difco) containing 50 μg/ml ampicillin and 250 μg/ml sodium azide (Sigma) to detect transconjugants.

Activity Assay:

Article Title: Characterization of Hemolytic Escherichia coli Strains in Ferrets: Recognition of Candidate Virulence Factor CNF1
Article Snippet: .. Bacterial sonicates or supernatants of seven ferret E. coli isolates and one nonpathogenic laboratory strain of E. coli without detectable cnf DNA homology or CNF activity, DH5α (Invitrogen, Carlsbad, Calif.), were evaluated for cytopathic effects on HeLa cell (CCL-2) monolayers. ..

Expressing:

Article Title: Specificity of the Type II Secretion Systems of Enterotoxigenic Escherichia coli and Vibrio cholerae for Heat-Labile Enterotoxin and Cholera Toxin ▿
Article Snippet: Plasmids were conjugated into V. cholerae by triparental mating as described previously , using DH5α (Invitrogen) containing the appropriate plasmid as the donor strain and MT616 carrying pRK2013 as the helper strain. .. Similar levels of expression of wild-type and mutant LT, LTB, or CTB constructs were detected after induction with a final concentration of 200 μM isopropyl-1-thio-β- d -galactopyranoside (IPTG; Sigma), except for the CTB[Q3K] construct, which required induction with 50 μM IPTG.

Article Title: A Novel, Molybdenum-Containing Methionine Sulfoxide Reductase Supports Survival of Haemophilus influenzae in an In vivo Model of Infection
Article Snippet: Construction of a TorZ Overexpression Plasmid A pProexHtb (Invitrogen) based protein expression plasmid containing the torZ gene without the region coding for the signal peptide was constructed using primers HIbisC-SP_pPro_Bam_F and HIbisC_pPro_Xba_R ( Table ) and the restriction enzyme sites introduced during high fidelity PCR. .. The resulting ligation products were transformed into DH5α (Life Technologies) to obtain pProex HItorZ -SP, which was verified by DNA sequencing.

Modification:

Article Title: Construction and high-throughput phenotypic screening of Zymoseptoria tritici over-expression strains
Article Snippet: .. All Gateway®Entry and modified destination vectors were propagated in DH5α (Invitrogen, UK). .. The kanamycin sensitive A. tumefaciens strain EHA105 ( ) was used for Z. tritici transformation.

Transformation Assay:

Article Title: A Novel, Molybdenum-Containing Methionine Sulfoxide Reductase Supports Survival of Haemophilus influenzae in an In vivo Model of Infection
Article Snippet: .. The resulting ligation products were transformed into DH5α (Life Technologies) to obtain pProex HItorZ -SP, which was verified by DNA sequencing. .. Optimal expression of the recombinant TorZ (rTorZ) protein was obtained using pProex HItorZ -SP in E. coli DH5α and LB medium supplemented with 1 mM sodium molybdate and 100 μg/mL ampicillin.

Article Title: KPC-4 Is Encoded within a Truncated Tn4401 in an IncL/M Plasmid, pNE1280, Isolated from Enterobacter cloacae and Serratia marcescens
Article Snippet: .. Plasmid DNA was transformed into E. coli ElectroMAX Stbl4 or DH5α (Invitrogen, Carlsbad, CA) using heat shock and the manufacturer's recommendations. .. Conjugation experiments were performed by inoculating equal amounts (105 CFU) of the donor (either E. cloacae 1623 or S. marcescens 1638; both ampicillin resistant and sodium azide susceptible) and the recipient ( E. coli J53; ampicillin susceptible and sodium azide resistant) ( , ) onto a nitrocellulose filter (Millipore; 0.025-μm VSWP) placed on a brain heart infusion agar (Difco) plate.

Article Title: Construction and high-throughput phenotypic screening of Zymoseptoria tritici over-expression strains
Article Snippet: In this IPO323 derivative, the Ku70 gene (Mycgr3G85040) of Z. tritici has been replaced with a G418 cassette using A. tumefaciens mediated transformation. .. All Gateway®Entry and modified destination vectors were propagated in DH5α (Invitrogen, UK).

Article Title: Three DUF1996 Proteins Localize in Vacuoles and Function in Fungal Responses to Multiple Stresses and Metal Ions
Article Snippet: E. coli Top10 and DH5α from Invitrogen (Shanghai, China) were cultured in Luria-Bertani medium plus kanamycin (100 μg/ml) or ampicillin (100 μg/ml) for plasmid propagation. .. Agrobacterium tumefaciens AGL-1, cultivated in YEB medium , was used as a T-DNA donor for fungal transformation.

Over Expression:

Article Title: A Novel, Molybdenum-Containing Methionine Sulfoxide Reductase Supports Survival of Haemophilus influenzae in an In vivo Model of Infection
Article Snippet: Paragraph title: Construction of a TorZ Overexpression Plasmid ... The resulting ligation products were transformed into DH5α (Life Technologies) to obtain pProex HItorZ -SP, which was verified by DNA sequencing.

Derivative Assay:

Article Title: Transcriptional and post-transcriptional regulation of PenA β-lactamase in acquired Burkholderia pseudomallei β-lactam resistance
Article Snippet: Bacterial strains and growth Escherichia coli strains used in this study were DH5α (ThermoFisher Scientific, Waltham, MA) and XL1 Blue (Agilent Technologies, Santa Clara, CA) for routine cloning and RHO3 for conjugation . .. Strains derived from Bp1651 and Bp82.27 are listed in Tables and , respectively.

Article Title: Transcriptional and post-transcriptional regulation of PenA β-lactamase in acquired Burkholderia pseudomallei β-lactam resistance
Article Snippet: Escherichia coli strains used in this study were DH5α (ThermoFisher Scientific, Waltham, MA) and XL1 Blue (Agilent Technologies, Santa Clara, CA) for routine cloning and RHO3 for conjugation . .. Strains derived from Bp1651 and Bp82.27 are listed in Tables and , respectively.

Hybridization:

Article Title: KPC-4 Is Encoded within a Truncated Tn4401 in an IncL/M Plasmid, pNE1280, Isolated from Enterobacter cloacae and Serratia marcescens
Article Snippet: The subsequent hybridization and development of the Southern blots were performed as described in the manufacturer's DIG application manual (Roche). .. Plasmid DNA was transformed into E. coli ElectroMAX Stbl4 or DH5α (Invitrogen, Carlsbad, CA) using heat shock and the manufacturer's recommendations.

Conjugation Assay:

Article Title: Transcriptional and post-transcriptional regulation of PenA β-lactamase in acquired Burkholderia pseudomallei β-lactam resistance
Article Snippet: .. Bacterial strains and growth Escherichia coli strains used in this study were DH5α (ThermoFisher Scientific, Waltham, MA) and XL1 Blue (Agilent Technologies, Santa Clara, CA) for routine cloning and RHO3 for conjugation . ..

Article Title: Transcriptional and post-transcriptional regulation of PenA β-lactamase in acquired Burkholderia pseudomallei β-lactam resistance
Article Snippet: .. Escherichia coli strains used in this study were DH5α (ThermoFisher Scientific, Waltham, MA) and XL1 Blue (Agilent Technologies, Santa Clara, CA) for routine cloning and RHO3 for conjugation . ..

Article Title: KPC-4 Is Encoded within a Truncated Tn4401 in an IncL/M Plasmid, pNE1280, Isolated from Enterobacter cloacae and Serratia marcescens
Article Snippet: Plasmid DNA was transformed into E. coli ElectroMAX Stbl4 or DH5α (Invitrogen, Carlsbad, CA) using heat shock and the manufacturer's recommendations. .. Conjugation experiments were performed by inoculating equal amounts (105 CFU) of the donor (either E. cloacae 1623 or S. marcescens 1638; both ampicillin resistant and sodium azide susceptible) and the recipient ( E. coli J53; ampicillin susceptible and sodium azide resistant) ( , ) onto a nitrocellulose filter (Millipore; 0.025-μm VSWP) placed on a brain heart infusion agar (Difco) plate.

Southern Blot:

Article Title: KPC-4 Is Encoded within a Truncated Tn4401 in an IncL/M Plasmid, pNE1280, Isolated from Enterobacter cloacae and Serratia marcescens
Article Snippet: Southern blot analysis ( ) was performed using a bla KPC-4 DNA probe generated using KPC forward and reverse; DNA probes were subsequently labeled with digoxigenin-labeled dUTP (Roche). .. Plasmid DNA was transformed into E. coli ElectroMAX Stbl4 or DH5α (Invitrogen, Carlsbad, CA) using heat shock and the manufacturer's recommendations.

Ligation:

Article Title: A Novel, Molybdenum-Containing Methionine Sulfoxide Reductase Supports Survival of Haemophilus influenzae in an In vivo Model of Infection
Article Snippet: .. The resulting ligation products were transformed into DH5α (Life Technologies) to obtain pProex HItorZ -SP, which was verified by DNA sequencing. .. Optimal expression of the recombinant TorZ (rTorZ) protein was obtained using pProex HItorZ -SP in E. coli DH5α and LB medium supplemented with 1 mM sodium molybdate and 100 μg/mL ampicillin.

Protease Inhibitor:

Article Title: Alteration in the cavity size adjacent to the active site of RB69 DNA polymerase changes its conformational dynamics
Article Snippet: Escherichia coli strain BL21 GOLD (DE3) and DH5α were from Invitrogen. .. The dNTP stock solutions (100 mM) and EDTA-free protease inhibitor cocktail tablets were from Roche.

Cell Culture:

Article Title: Aggregative Adherence and Intestinal Colonization by Enteroaggregative Escherichia coli Are Produced by Interactions among Multiple Surface Factors
Article Snippet: For maintenance and molecular biology procedures, bacterial strains were routinely cultured in Luria broth (LB) or LB agar and maintained in LB-glycerol (1:1) at −80°C. .. We used E. coli K-12 strains TOP10 and DH5α (Invitrogen) as host strains for cloned genes unless otherwise indicated.

Article Title: Three DUF1996 Proteins Localize in Vacuoles and Function in Fungal Responses to Multiple Stresses and Metal Ions
Article Snippet: .. E. coli Top10 and DH5α from Invitrogen (Shanghai, China) were cultured in Luria-Bertani medium plus kanamycin (100 μg/ml) or ampicillin (100 μg/ml) for plasmid propagation. .. Agrobacterium tumefaciens AGL-1, cultivated in YEB medium , was used as a T-DNA donor for fungal transformation.

Generated:

Article Title: KPC-4 Is Encoded within a Truncated Tn4401 in an IncL/M Plasmid, pNE1280, Isolated from Enterobacter cloacae and Serratia marcescens
Article Snippet: Southern blot analysis ( ) was performed using a bla KPC-4 DNA probe generated using KPC forward and reverse; DNA probes were subsequently labeled with digoxigenin-labeled dUTP (Roche). .. Plasmid DNA was transformed into E. coli ElectroMAX Stbl4 or DH5α (Invitrogen, Carlsbad, CA) using heat shock and the manufacturer's recommendations.

DNA Sequencing:

Article Title: A Novel, Molybdenum-Containing Methionine Sulfoxide Reductase Supports Survival of Haemophilus influenzae in an In vivo Model of Infection
Article Snippet: .. The resulting ligation products were transformed into DH5α (Life Technologies) to obtain pProex HItorZ -SP, which was verified by DNA sequencing. .. Optimal expression of the recombinant TorZ (rTorZ) protein was obtained using pProex HItorZ -SP in E. coli DH5α and LB medium supplemented with 1 mM sodium molybdate and 100 μg/mL ampicillin.

Isolation:

Article Title: Aggregative Adherence and Intestinal Colonization by Enteroaggregative Escherichia coli Are Produced by Interactions among Multiple Surface Factors
Article Snippet: EAEC strain 042 was originally isolated in Peru and elicited diarrhea in three of five adult volunteers during a human challenge study ( ). .. We used E. coli K-12 strains TOP10 and DH5α (Invitrogen) as host strains for cloned genes unless otherwise indicated.

Article Title: KPC-4 Is Encoded within a Truncated Tn4401 in an IncL/M Plasmid, pNE1280, Isolated from Enterobacter cloacae and Serratia marcescens
Article Snippet: Plasmid was isolated from E. cloacae and E. coli using either the method of Kado and Liu ( ) or the Qiagen (Valencia, CA) Large Construct Kit following the manufacturer's recommendations. .. Plasmid DNA was transformed into E. coli ElectroMAX Stbl4 or DH5α (Invitrogen, Carlsbad, CA) using heat shock and the manufacturer's recommendations.

Polymerase Chain Reaction:

Article Title: A Novel, Molybdenum-Containing Methionine Sulfoxide Reductase Supports Survival of Haemophilus influenzae in an In vivo Model of Infection
Article Snippet: Construction of a TorZ Overexpression Plasmid A pProexHtb (Invitrogen) based protein expression plasmid containing the torZ gene without the region coding for the signal peptide was constructed using primers HIbisC-SP_pPro_Bam_F and HIbisC_pPro_Xba_R ( Table ) and the restriction enzyme sites introduced during high fidelity PCR. .. The resulting ligation products were transformed into DH5α (Life Technologies) to obtain pProex HItorZ -SP, which was verified by DNA sequencing.

Recombinant:

Article Title: A Novel, Molybdenum-Containing Methionine Sulfoxide Reductase Supports Survival of Haemophilus influenzae in an In vivo Model of Infection
Article Snippet: The resulting ligation products were transformed into DH5α (Life Technologies) to obtain pProex HItorZ -SP, which was verified by DNA sequencing. .. Optimal expression of the recombinant TorZ (rTorZ) protein was obtained using pProex HItorZ -SP in E. coli DH5α and LB medium supplemented with 1 mM sodium molybdate and 100 μg/mL ampicillin.

Pulsed-Field Gel:

Article Title: KPC-4 Is Encoded within a Truncated Tn4401 in an IncL/M Plasmid, pNE1280, Isolated from Enterobacter cloacae and Serratia marcescens
Article Snippet: Pulsed-field gel electrophoresis (PFGE) was performed according to the standard Pulsenet protocol for Escherichia coli , as described by Ribot et al. ( ). .. Plasmid DNA was transformed into E. coli ElectroMAX Stbl4 or DH5α (Invitrogen, Carlsbad, CA) using heat shock and the manufacturer's recommendations.

Mutagenesis:

Article Title: Specificity of the Type II Secretion Systems of Enterotoxigenic Escherichia coli and Vibrio cholerae for Heat-Labile Enterotoxin and Cholera Toxin ▿
Article Snippet: Plasmids were conjugated into V. cholerae by triparental mating as described previously , using DH5α (Invitrogen) containing the appropriate plasmid as the donor strain and MT616 carrying pRK2013 as the helper strain. .. Similar levels of expression of wild-type and mutant LT, LTB, or CTB constructs were detected after induction with a final concentration of 200 μM isopropyl-1-thio-β- d -galactopyranoside (IPTG; Sigma), except for the CTB[Q3K] construct, which required induction with 50 μM IPTG.

CtB Assay:

Article Title: Specificity of the Type II Secretion Systems of Enterotoxigenic Escherichia coli and Vibrio cholerae for Heat-Labile Enterotoxin and Cholera Toxin ▿
Article Snippet: Plasmids were conjugated into V. cholerae by triparental mating as described previously , using DH5α (Invitrogen) containing the appropriate plasmid as the donor strain and MT616 carrying pRK2013 as the helper strain. .. Similar levels of expression of wild-type and mutant LT, LTB, or CTB constructs were detected after induction with a final concentration of 200 μM isopropyl-1-thio-β- d -galactopyranoside (IPTG; Sigma), except for the CTB[Q3K] construct, which required induction with 50 μM IPTG.

Labeling:

Article Title: KPC-4 Is Encoded within a Truncated Tn4401 in an IncL/M Plasmid, pNE1280, Isolated from Enterobacter cloacae and Serratia marcescens
Article Snippet: Southern blot analysis ( ) was performed using a bla KPC-4 DNA probe generated using KPC forward and reverse; DNA probes were subsequently labeled with digoxigenin-labeled dUTP (Roche). .. Plasmid DNA was transformed into E. coli ElectroMAX Stbl4 or DH5α (Invitrogen, Carlsbad, CA) using heat shock and the manufacturer's recommendations.

Purification:

Article Title: Alteration in the cavity size adjacent to the active site of RB69 DNA polymerase changes its conformational dynamics
Article Snippet: Materials All oligonucleotides were synthesized at the Keck facilities (Yale University) and purified via polyacrylamide gel electrophoresis. .. Escherichia coli strain BL21 GOLD (DE3) and DH5α were from Invitrogen.

Polyacrylamide Gel Electrophoresis:

Article Title: Alteration in the cavity size adjacent to the active site of RB69 DNA polymerase changes its conformational dynamics
Article Snippet: Materials All oligonucleotides were synthesized at the Keck facilities (Yale University) and purified via polyacrylamide gel electrophoresis. .. Escherichia coli strain BL21 GOLD (DE3) and DH5α were from Invitrogen.

Plasmid Preparation:

Article Title: Invasion of Eukaryotic Cells by Borrelia burgdorferi Requires ?1 Integrins and Src Kinase Activity ▿ Integrins and Src Kinase Activity ▿ ‡
Article Snippet: .. Escherichia coli strains TOP10 and DH5α (Invitrogen, Carlsbad, CA) were used for plasmid propagation under the following antibiotic selection: 5 μg/ml gentamicin, 50 μg/ml kanamycin, 100 μg/ml spectinomycin, and 15 μg/ml chloramphenicol. ..

Article Title: Specificity of the Type II Secretion Systems of Enterotoxigenic Escherichia coli and Vibrio cholerae for Heat-Labile Enterotoxin and Cholera Toxin ▿
Article Snippet: .. Plasmids were conjugated into V. cholerae by triparental mating as described previously , using DH5α (Invitrogen) containing the appropriate plasmid as the donor strain and MT616 carrying pRK2013 as the helper strain. .. Selection of transconjugants was carried out on LB agar plates supplemented with kanamycin and ampicillin.

Article Title: Deletion of ptn1, a PTEN/TEP1 Orthologue, in Ustilago maydis Reduces Pathogenicity and Teliospore Development
Article Snippet: .. Escherichia coli strains TOP10 and DH5α (Invitrogen/Thermo Fisher) were used for all cloning and plasmid maintenance. ..

Article Title: A Novel, Molybdenum-Containing Methionine Sulfoxide Reductase Supports Survival of Haemophilus influenzae in an In vivo Model of Infection
Article Snippet: Paragraph title: Construction of a TorZ Overexpression Plasmid ... The resulting ligation products were transformed into DH5α (Life Technologies) to obtain pProex HItorZ -SP, which was verified by DNA sequencing.

Article Title: KPC-4 Is Encoded within a Truncated Tn4401 in an IncL/M Plasmid, pNE1280, Isolated from Enterobacter cloacae and Serratia marcescens
Article Snippet: .. Plasmid DNA was transformed into E. coli ElectroMAX Stbl4 or DH5α (Invitrogen, Carlsbad, CA) using heat shock and the manufacturer's recommendations. .. Conjugation experiments were performed by inoculating equal amounts (105 CFU) of the donor (either E. cloacae 1623 or S. marcescens 1638; both ampicillin resistant and sodium azide susceptible) and the recipient ( E. coli J53; ampicillin susceptible and sodium azide resistant) ( , ) onto a nitrocellulose filter (Millipore; 0.025-μm VSWP) placed on a brain heart infusion agar (Difco) plate.

Article Title: Three DUF1996 Proteins Localize in Vacuoles and Function in Fungal Responses to Multiple Stresses and Metal Ions
Article Snippet: .. E. coli Top10 and DH5α from Invitrogen (Shanghai, China) were cultured in Luria-Bertani medium plus kanamycin (100 μg/ml) or ampicillin (100 μg/ml) for plasmid propagation. .. Agrobacterium tumefaciens AGL-1, cultivated in YEB medium , was used as a T-DNA donor for fungal transformation.

Selection:

Article Title: Invasion of Eukaryotic Cells by Borrelia burgdorferi Requires ?1 Integrins and Src Kinase Activity ▿ Integrins and Src Kinase Activity ▿ ‡
Article Snippet: .. Escherichia coli strains TOP10 and DH5α (Invitrogen, Carlsbad, CA) were used for plasmid propagation under the following antibiotic selection: 5 μg/ml gentamicin, 50 μg/ml kanamycin, 100 μg/ml spectinomycin, and 15 μg/ml chloramphenicol. ..

Article Title: Aggregative Adherence and Intestinal Colonization by Enteroaggregative Escherichia coli Are Produced by Interactions among Multiple Surface Factors
Article Snippet: Antibiotics, including ampicillin (100 µg/ml), chloramphenicol (30 µg/ml), tetracycline (25 µg/ml), and neomycin (50 µg/ml), were added for selection when required. .. We used E. coli K-12 strains TOP10 and DH5α (Invitrogen) as host strains for cloned genes unless otherwise indicated.

Article Title: Specificity of the Type II Secretion Systems of Enterotoxigenic Escherichia coli and Vibrio cholerae for Heat-Labile Enterotoxin and Cholera Toxin ▿
Article Snippet: Plasmids were conjugated into V. cholerae by triparental mating as described previously , using DH5α (Invitrogen) containing the appropriate plasmid as the donor strain and MT616 carrying pRK2013 as the helper strain. .. Selection of transconjugants was carried out on LB agar plates supplemented with kanamycin and ampicillin.

Concentration Assay:

Article Title: Specificity of the Type II Secretion Systems of Enterotoxigenic Escherichia coli and Vibrio cholerae for Heat-Labile Enterotoxin and Cholera Toxin ▿
Article Snippet: Plasmids were conjugated into V. cholerae by triparental mating as described previously , using DH5α (Invitrogen) containing the appropriate plasmid as the donor strain and MT616 carrying pRK2013 as the helper strain. .. Similar levels of expression of wild-type and mutant LT, LTB, or CTB constructs were detected after induction with a final concentration of 200 μM isopropyl-1-thio-β- d -galactopyranoside (IPTG; Sigma), except for the CTB[Q3K] construct, which required induction with 50 μM IPTG.

Article Title: A Novel, Molybdenum-Containing Methionine Sulfoxide Reductase Supports Survival of Haemophilus influenzae in an In vivo Model of Infection
Article Snippet: The resulting ligation products were transformed into DH5α (Life Technologies) to obtain pProex HItorZ -SP, which was verified by DNA sequencing. .. IPTG was added to a final concentration of 100 μM and the cultures incubated at 30°C, 200 rpm overnight before harvesting of cells by centrifugation (3000 × g , 4°C, 10 min).

Article Title: Functional replacement of the endogenous tyrosyl-tRNA synthetase-tRNATyr pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion
Article Snippet: Strains, growth conditions, growth rate, reagents and non-natural amino acids Escherichia coli strains TOP10, BL21(DE3) and DH5α were purchased from Invitrogen, Novagen and Toyobo (Tokyo, Japan), respectively. .. Kanamycin (Km) (30 µg/ml), chloramphenicol (Cm) (34 µg/ml), ampicillin (Amp) (10 µg/ml), Zeocin (25 µg/ml), l -arabinose (0.2%, w/v) and d -glucose (0.4%, w/v) were added to LB media as indicated, except that Cm was added at a concentration of 25 µg/ml when non-natural amino acids were incorporated into proteins.

Article Title: Analysis of the Behavior of eryC Mutants of Brucella suis Attenuated in Macrophages
Article Snippet: .. The Escherichia coli strains CC118λpir ( ) and DH5α (Invitrogen, Cergy Pontoise, France) were used as host strains for the cloning experiments with the erythritol gene of B. suis ; they were grown in Luria-Bertani broth in the presence of kanamycin or ampicillin at a concentration of 50 μg/ml when appropriate. .. Constructions were performed using plasmids pUC4K, pUC18 (both Amersham Biosciences, Orsay France), pGEM-T Easy (Promega, Charbonnières, France), pBBR1MCS , and pCVD442 ( ).

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    Thermo Fisher subcloning efficiency dh5α competent cells
    Subcloning Efficiency Dh5α Competent Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MultiShot StripWell DH5α T1R chemically competent E coli cells are cloning competent cells that are packaged in a rack containing 12 strips of 8 wells to increase productivity for medium
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