Recoverease dna isolation kit

The gpt assay was performed as described previously [31 (link)]. Briefly, DNA was extracted from lung tissue by using a RecoverEase DNA Isolation Kit (Agilent Technologies, Santa Clara, CA, USA) and lambda EG10 phages were rescued by using Transpack Packaging Extract (Agilent Technologies). E. coli YG6020 were infected with the rescued phages, inoculated to M9 salt plates containing chloramphenicol (Cm) and 6-thioguanine (6-TG), and then incubated for 72–90 h at 37 °C, which enabled selection of colonies harboring a plasmid carrying both the gene for chloramphenicol acetyltransferase as well as a mutated gpt gene. Isolates exhibiting the 6-TG-resistant phenotype were cultured overnight at 37 °C in Luria–Bertani (LB) broth containing 25 μg/mL Cm, harvested by centrifugation (7000 rpm, 10 min), and then stored at − 80 °C.

High molecular weight DNA isolation using the RecoverEase™ DNA isolation kit followed the manufacturer's instructions (Agilent Technologies, Santa Clara, CA) as described previously [34] (link). Lambda phage shuttle vectors harboring the bacterial lacI gene were recovered from high molecular weight DNA samples using Agilent Technologies' Transpack in vitro packaging extracts. Packaged phage were mixed with E. coli SCS-8 cells and added to top agarose containing 5-bromo-4-chloro-3-indoyl-betagalactopyranoside (X-gal) and plated on NZY agar trays. Plaque-forming units (pfus) were counted after incubation overnight at 37°C. Putative blue mutant plaques were visually identified and confirmed by coring and plating again. Mutant frequency was determined by dividing the number of confirmed mutant plaques by the total number of pfus obtained for the sample.

High molecular weight genomic DNA from the colonic, hepatic, and pulmonary organoids was extracted using RecoverEase DNA Isolation Kit (Agilent Technology, United States), according to the manufacturer’s instructions. Lambda EG10 phages were rescued using Transpack Packaging Extract (Stratagene, La Jolla, CA, United States). The gpt mutation assay was performed according to previously described methods (Fukai et al., 2018 (link)). Briefly, E. coli YG6020 was infected with the phage and spread on M9 salt plates containing chloramphenicol (Cm) and 6-thioguanine (6-TG) and incubated for 72 h at 37°C to select colonies with a plasmid carrying the gene encoding chloramphenicol acetyltransferase, as well as mutated gpt. The 6-TG–resistant isolates were cultured overnight at 37°C in LB broth containing 25 mg/ml of Cm, harvested by centrifugation (7,000 rpm, 10 min), and stored at −80°C. Mutational spectra of 6-TG coding sequences were determined using PCR and direct sequencing, and a 739-bp DNA fragment containing gpt was amplified by PCR as described previously (Fukai et al., 2018 (link)). Sequence analysis was performed using Eurofins Genomics software (Tokyo, Japan).

The gpt mutation assay was performed as described previously^{12 (link))}. Briefly, DNA was extracted from
the small intestine mucosa by means of a RecoverEase DNA Isolation Kit (Agilent
Technologies, Santa Clara, CA, USA), and lambda EG10 phages were recovered with Transpack
Packaging Extract (Agilent Technologies). Escherichia coli YG6020 were
infected with the recovered phages, plated on M9 salt plates containing chloramphenicol
(Cm) and 6-thioguanine (6-TG), and then incubated for 72–90 h at 37°C. This incubation
enabled selection of colonies harboring a plasmid carrying both the gene for
chloramphenicol acetyltransferase and a mutated gpt gene.
gpt-Mutant frequency was calculated by dividing the number of mutated
colonies growing on agar plates containing Cm and 6-TG by the number of colonies growing
on agar plates containing Cm alone. The mutants exhibiting the 6-TG-resistant phenotype
were cultured overnight at 37°C in Luria–Bertani broth containing 25 µg/mL Cm, harvested
by centrifugation (7000 rpm, 10 min), and then stored at −80°C. A 739-bp DNA fragment
containing gpt was amplified by means of the polymerase chain reaction
and sequenced as described previously^{12 (link),13 (link))}.