pTALBamHI DNA was partially digested with the restriction enzyme MscI as follow: 2 μg pTALBamHI plasmid DNA, 5 u of MscI (New England BioLabs), and 2 μl of digestion buffer were mixed; the final reaction volume was adjusted to 20 μl with water, and digestions were performed at 37 °C for a period of time and stopped by heating at 65 °C for 10 minutes and supplementing with 3 μl of 10% SDS-containing loading dye (Takara). The DNA fragments were separated by eletrophoresis in agarose gel and the about 1-kb DNA band was sliced out from the gel and purified using QIAquick Gel Extraction Kit. The purified DNA fragments were vacuum-concentrated, and 20 ng of them was used for the ligation with 15 ng of pCC2FOS vector linearized with the restriction enzyme Eco72 I (Epicentre). The ligation products were electroporated into the E. coli strain EPI300-T1R (Epicenter) and the transformants were selected on chloramphenicol-containing LB medium supplemented with IPTG (120 μg/ml) and X-gal (80 μg/ml). The copy number induction of the pCC2FOS-MscI clones were performed prior to plasmid extraction using CopyControl™ Induction Solution (Epicentre) according to the provided manual.
Copycontrol induction solution
Manufactured by Illumina
Sourced in United States
The CopyControl Induction Solution is a laboratory product designed to facilitate the regulation of plasmid copy number in bacterial cultures. It provides a controlled environment for the modulation of plasmid replication, allowing for the precise management of DNA copy numbers during experimentation or production processes.
Automatically generated - may contain errors
Lab products found in correlation
Alliance 2690 hplc, by Waters Corporation (4 mentions)
Heto powerdry ll1500 freeze dryer, by Thermo Fisher Scientific (4 mentions)
Ghp bulk acrodisc 13, by Pall Corporation (4 mentions)
Epi300 t1r, by Illumina (1 mentions)
Qiaprep kit, by Qiagen (1 mentions)
Fosmidmax dna purification kit, by Illumina (1 mentions)
Fast link dna ligase kit, by Illumina (1 mentions)
Qiaprep spin miniprep kit, by Qiagen (1 mentions)
Zr bac dna miniprep kit, by Zymo Research (1 mentions)
9 protocols using copycontrol induction solution
1
Constructing a pCC2FOS Clone Library
2
Amplification and Characterization of Bacterial Metabolites
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Example 6
E. coli cells carrying the subclone pFosS1A and control (empty vector) were grown overnight at 37° C. in LB-broth containing chloramphenicol. These cultures were used as inocula for the copy number amplification procedure. One volume of these cultures was added to 10 volumes of fresh LB+chloramphenicol and 1/100 of CopyControl Induction Solution (Epicentre) was added to the media to induce clones to high copy number. After vigorous shaking of cultures at 37° C. for 20 h, the supernatant was separated from bacterial cells by centrifugation for 20 min at 3040×g at 4° C. The supernatant was freeze-dried by Heto PowerDry LL1500 freeze dryer (ThermoElectron, Mukarov, Czech Republic). The freeze-dried broth was weighed and 50 mg was dissolved to 2 ml of distilled water. The material was kept in ultrasonicator for 20 minutes, centrifuged for 10 minutes, filtered (GHP Bulk Acrodisc 13, Pall Life Sciences) and fractioned by semi preparative High Pressure Liquid Chromatography (HPLC). The fractions were then tested for antibacterial activity by the 96-well plate standard method using S. aureus as the test strain. Supernatants of subclone pFosS1A and control were analyzed for small molecules by Alliance 2690 HPLC (Waters, Milford, Mass., USA) combined with Micromass LCT Time-of-flight mass spectrometry (TOFMS) (Micromass, Altrincham, UK).
3
Amplification and Purification of Plasmid DNA
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Example 6
E. coli cells carrying the subclone pFosS1A and control (empty vector) were grown overnight at 37° C. in LB-broth containing chloramphenicol. These cultures were used as inocula for the copy number amplification procedure. One volume of these cultures was added to 10 volumes of fresh LB+chloramphenicol and 1/100 of CopyControl Induction Solution (Epicentre) was added to the media to induce clones to high copy number. After vigorous shaking of cultures at 37° C. for 20 h, the supernatant was separated from bacterial cells by centrifugation for 20 min at 3040× g at 4° C. The supernatant was freeze-dried by Heto PowerDry LL1500 freeze dryer (ThermoElectron, Mukarov, Czech Republic). The freeze-dried broth was weighed and 50 mg was dissolved to 2 ml of distilled water. The material was kept in ultrasonicator for 20 minutes, centrifuged for 10 minutes, filtered (GHP Bulk Acrodisc 13, Pall Life Sciences) and fractioned by semi preparative High Pressure Liquid Chromatography (HPLC). The fractions were then tested for antibacterial activity by the 96-well plate standard method using S. aureus as the test strain. Supernatants of subclone pFosS1A and control were analyzed for small molecules by Alliance 2690 HPLC (Waters, Milford, Mass., USA) combined with Micromass LCT Time-of-flight mass spectrometry (TOFMS) (Micromass, Altrincham, UK).
4
E. coli Copy Number Amplification
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Example 6
E. coli cells carrying the subclone pFosS1A and control (empty vector) were grown overnight at 37° C. in LB-broth containing chloramphenicol. These cultures were used as inocula for the copy number amplification procedure. One volume of these cultures was added to 10 volumes of fresh LB+chloramphenicol and 1/100 of CopyControl Induction Solution (Epicentre) was added to the media to induce clones to high copy number. After vigorous shaking of cultures at 37° C. for 20 h, the supernatant was separated from bacterial cells by centrifugation for 20 min at 3040×g at 4° C. The supernatant was freeze-dried by Heto PowerDry LL1500 freeze dryer (ThermoElectron, Mukarov, Czech Republic). The freeze-dried broth was weighed and 50 mg was dissolved to 2 ml of distilled water. The material was kept in ultrasonicator for 20 minutes, centrifuged for 10 minutes, filtered (GHP Bulk Acrodisc 13, Pall Life Sciences) and fractioned by semi preparative High Pressure Liquid Chromatography (HPLC). The fractions were then tested for antibacterial activity by the 96-well plate standard method using S. aureus as the test strain. Supernatants of subclone pFosS1A and control were analyzed for small molecules by Alliance 2690 HPLC (Waters, Milford, Mass., USA) combined with Micromass LCT Time-of-flight mass spectrometry (TOFMS) (Micromass, Altrincham, UK).
5
Analyzing Antibiotic Resistance Genomic Context
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To better understand the genomic context of twelve distinct clones conferring AR, we sequenced the entire fosmid in each using Pacific Biosciences (PacBio) RS technology. DNA was prepared using the QIAprep kit (Qiagen) from 10 ml of bacterial culture in LB supplemented with chloramphenicol and 1× CopyControl induction solution (Epicentre). From each fosmid clone, 300 ng of DNA was pooled into a single reaction mixture and submitted to the Yale high-throughput sequencing center (http://medicine.yale.edu/keck/ycga ). Assembly was performed using Celera (51 (link)) and trimmed for fosmid vector sequence using the ContaminationTrimmer.py command (https://github.com/PacificBiosciences/HBAR-DTK) . The resulting scaffolds were assigned to the fosmids by performing a BLASTN search for the resistance genes and end sequences generated by Sanger sequencing of the fosmid vector using the primers recommended by the manufacturer. The assembly of 5,037 high-quality reads resulted in 45 scaffolds with an average coverage of 7.88×. Twelve of these scaffolds contained the expected antibiotic resistance genes.
6
E. coli Copy Number Amplification
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Example 6
E. coli cells carrying the subclone pFosS1A and control (empty vector) were grown overnight at 37° C. in LB-broth containing chloramphenicol. These cultures were used as inocula for the copy number amplification procedure. One volume of these cultures was added to 10 volumes of fresh LB+chloramphenicol and 1/100 of CopyControl Induction Solution (Epicentre) was added to the media to induce clones to high copy number. After vigorous shaking of cultures at 37° C. for 20 h, the supernatant was separated from bacterial cells by centrifugation for 20 min at 3040×g at 4° C. The supernatant was freeze-dried by Heto PowerDry LL1500 freeze dryer (ThermoElectron, Mukarov, Czech Republic). The freeze-dried broth was weighed and 50 mg was dissolved to 2 ml of distilled water. The material was kept in ultrasonicator for 20 minutes, centrifuged for 10 minutes, filtered (GHP Bulk Acrodisc 13, Pall Life Sciences) and fractioned by semi preparative High Pressure Liquid Chromatography (HPLC). The fractions were then tested for antibacterial activity by the 96-well plate standard method using S. aureus as the test strain. Supernatants of subclone pFosS1A and control were analyzed for small molecules by Alliance 2690 HPLC (Waters, Milford, Mass., USA) combined with Micromass LCT Time-of-flight mass spectrometry (TOFMS) (Micromass, Altrincham, UK).
7
Lipolytic Fosmid Clone Sequencing
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The fosmid clones which showed lipolytic activity were grown overnight in liquid LB medium, supplemented with 12.5 μg/mL chloramphenicol and Copy Control Induction solution (Epicentre, WI, USA) to induce the clone to high-copy number. Fosmid DNA of each culture was extracted using FosmidMAX™ DNA Purification Kit (Epicentre, WI, USA). One microgram of the fosmid DNA was sequenced using Illumina PE150 in the Novogene company (London, UK). A total of 5,176,886 reads with a read size of 150 bp were generated. Reads with ambiguous bases (“Ns”), sequence duplicates and low-complexity sequences with score quality value > 25 were removed using PRINSEQ (Schmieder and Edwards 2011a (link)). Removal of the readings corresponding to the pCC2FOS cloning vector (Genbank accession EU140752.1) and the genome of Escherichia coli (NC_000913) was undertaken using standalone Deconseq (version 0.4.3) with 90% coverage and 94% identity filtering options (Schmieder and Edwards 2011b (link)). Remaining reads were then assembled using Spades software (Bankevich et al. 2012 (link)).
8
Fosmid Extraction for Metagenomics
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Extraction of fosmids containing metagenomic DNA: 5 ml of bacterial culture was grown overnight with 12.5 μg/ml Cm. One millilitre of culture was used to inoculate 4 ml of fresh LB broth. To this, 5 μl of 1000× Copy Control Induction solution (Epicentre Biotechnologies) and 12.5 μg/ml Cm were added. The mixture was incubated at 37°C for 5 h with vigorous shaking (200–250 rpm) to ensure maximum aeration. Cells were harvested from the whole 5 ml of induced culture by centrifuging at 2100 × g for 12 min. Qiagen QIAprep Spin mini-prep kit was used to extract fosmids as per manufacturer's instructions. PCR products were purified with a Qiagen PCR purification kit and digested with XbaI and PstI (Roche Applied Science) for mfsT and with SalI and XbaI for sdtR, followed by ligation using the FastLink DNA ligase kit (Epicentre Biotechnologies) to similarly digested plasmid pCI372. Electrocompetent E. coli MKH13 and E. coli EPI300 were transformed with the ligation mixture and plated on LB agar plates containing 20 μg/ml Cm for selection. Colony PCR was performed on all resistant transformants using primers across the multiple cloning site (MCS) of pCI372 to confirm the presence and size of the insert.
9
Fosmid DNA Extraction from Bacterial Culture
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Bacterial culture (5 ml) was brought into existence after the cultivation of 12.5 μg/ml Cm for a night. Near 1 ml of the culture was added to the broth with 4 ml fresh LB. Moreover, 5 μl of the 1000 copy control induction solution (Epicentre Biotechnologies), together with 12.5 μg/ml Cm, was put in the culture. The mixture was cultured at 37°C for 5 h with vigorous shaking (200–250 rpm) to ensure maximum aeration. All 5-ml stimulated cultures generated cells after the 1-min centrifugation at 12000 rpm. ZR BAC DNA Miniprep Kit (Zymo Research) was used to extract fosmid based on the manufacturer’s directions.
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