Fosmids were subjected to mutagenesis using the EZ-Tn5TM Insertion kit (Epicentre Biotechnologies), according to the manufacturer's instructions. The mutant mini-library was plated on LB medium containing 12.5 μg/ml chloramphenicol and 10 μg/ml tetracycline. The clones were handpicked into liquid media in 96-well plates, incubated overnight and subsequently replicated onto solid LB medium containing 12.5 μg/ml chloramphenicol and 0.01% arabinose to induce the fosmids to multiple copies. The plates were then incubated overnight at 37°C, followed by 2 further overnight incubations at 25°C and overlaid with the B. subtilis DSM10 as described above. Mutants that appeared to have lost the ability to inhibit growth were selected for further analysis. The insertion site of the EZ-Tn5 transposon was mapped using bidirectional sequencing with primers TET-1 FP-1 (5′ GGG TGC GCA TGA TCC TCT AGA GT 3′) and TET-1 RP-1 (5′ TAA ATT GCA CTG AAA TCT AGA AAT A 3′).
Ez tn5tm insertion kit
Manufactured by Illumina
The EZ-Tn5TM Insertion kit is a laboratory equipment product designed for transposon insertion, a process used in genetic research and sequencing. The kit contains the necessary reagents and components to enable targeted integration of DNA sequences into target samples. The core function of the EZ-Tn5TM Insertion kit is to facilitate this transposon insertion procedure.
Automatically generated - may contain errors
2 protocols using ez tn5tm insertion kit
1
Fosmid Mutagenesis and Screening
2
Generation of Neisseria gonorrhoeae Transposon Library
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N. gonorrhoeae N2009 (MS11, P+, Opa−, PorBIA, Camr, Ermr) was used for transposon library generation, since this strain was fully transformation competent and in addition carried the PorBIA derivative mediating PorBIA-dependent invasion (36 (link)). Genomic DNA was extracted using the NucleoSpin Tissue kit (Macherey-Nagel) and 0.5 μg genomic DNA was mutagenized with 0.12 pmol Tn5 transposon in vitro (EZ-Tn5TM Insertion Kit, Epicentre Biotechnologies) and then purified by phenol extraction and ethanol precipitation. The mutagenized DNA was mixed with 1 U T4 DNA polymerase (Fermentas), 2 nmol dNTPs and incubated at 11°C for 20 min followed by heat inactivation at 75°C for 10 min. After phenol extraction and ethanol precipitation, the mutagenized DNA was ligated by 5 U T4 DNA ligase (Fermentas) at 16°C overnight and finally precipitated for transformation. 0.1 μg mutagenized DNA was mixed with 50 μl suspension of N. gonorrhoeae N2009 (OD550nm = 0.32) and incubated for 24 h on a GC agar plate and then colonies were transferred to GC agar plates supplemented with kanamycin and incubated for 48 h at 37°C. This was repeated six times resulting in a library harboring about 100 000 individual colonies. The colonies were harvested in PPM medium, mixed with 100% glycerol to a final concentration of 25% (v/v) and then stored at −80°C.
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