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17 protocols using recoverease dna isolation kit

1

UV-Induced Mutagenesis in Transgenic Mice

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Big Blue® mice or related lacZ mice (MutaMouse) have been successfully used to study both UVA & UVB-induced mutagenesis [60 (link), 61 (link)]. For mutation studies, WT and Pparg-/-epi mice expressing the λLIZ transgene were anesthetized and treated with increasing doses of UVB. Control mice were not UVB treated. Five days later we euthanized the mice and excised the treated skin for epidermal DNA isolation. We waited 5 days before euthanasia to allow for both complete repair of CPD lesions and for clearance of mutations in non-cancer causing suprabasal cells through epidermal turnover (epidermal transit time = 3-5 days for hairless mouse epidermis) [62 (link), 63 (link)]. For DNA isolation, we separated the epidermis from the subcutaneous tissue using thermolysin as described above. Cells of the epidermis were isolated by trypsinization in 0.25% trypsin and the cellular nuclei DNA was isolated using the RecoverEase DNA Isolation Kit (Agilent Technologies). Samples were dialyzed in TE buffer using G Biosciences Tube-O-Dialyzer. The mutation frequency was determined using Agilent Transpack Packaging Extract and λSelect-cII Mutation Detection System for Big Blue Rodents.
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2

Intestinal DNA Extraction Protocol

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The gpt mutation assay was performed as previously reported [7 (link), 8 (link)]. Briefly, genomic DNA was extracted from the mucosa of the small intestine by using a RecoverEase DNA Isolation Kit (Agilent Technologies, Santa Clara, CA, USA).
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3

Extraction and Phage Recovery from Lung DNA

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High-molecular-weight genomic DNA was extracted from the lungs by using the RecoverEase DNA Isolation Kit (Agilent Technologies, Santa Clara, CA, USA). Lambda EG10 phages containing the gpt gene were recovered from the genomic DNA by using Transpack Packaging Extract (Agilent Technologies).
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4

Isolation of 6-TG-Resistant Mutants

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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.
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5

Genomic Sequencing of Taiwanese Insect

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Live specimens were observed in their native habitat in Miaoli County, Taiwan (120°39′E; 24°29′N) and a dozen live specimens were collected in July of 2017 and May of 2018 by JPL. Presented data is based on genome extraction of a single individual. Genomic DNA was extracted with the RecoverEase DNA isolation kit (Agilent Technologies) and size-selected for molecules of 40 kb or larger using a BluePippin (Sage Science) device. DNA was quantified with Life Technologies Qubit and Agilent Technologies Tapestation 4200. For library preparation 0.625 ng size-selected genomic DNA was used. The barcoded library was prepared with the Chromium gel bead and library kit (10× Genomics), and sequencing was performed with an Illumina Novaseq 6000 sequencer with 151 bp paired-end reads. All read pairs contain a 16-base barcode.
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6

Genomic DNA Extraction and Phage Packaging

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Liver tissue was pulverized in a mortar and pestle under liquid nitrogen and stored at −80°C. Genomic DNA was extracted from approximately 25 mg of tissue using the RecoverEase DNA Isolation Kit (Agilent Technologies, Santa Clara, CA). λ-EG10 phages were packaged in vitro from genomic DNA using Transpack Packaging Extract (Agilent Technologies) following the manufacturer's instructions.
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7

Bacterial Lacl Mutant Screening

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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.
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8

Isolation of High-Quality Genomic DNA

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Long genomic DNA was isolated from cell lines following a modified version of the RecoverEase DNA isolation kit (Agilent Technologies) protocol (https://www.agilent.com/cs/library/usermanuals/public/200600.pdf). Briefly, approximately 1 million cells were pelleted and lysed with 500 µL of lysis buffer. After a 10-min incubation at 4°C, 20 µL RNace-It ribonuclease cocktail in 4 mL digestion buffer was added directly to the lysed cells and incubated on a 50°C heat block. After 5 min, 4.5 mL Proteinase K solution (∼1.1 mg/mL Proteinase K, 0.56% SDS, and 0.89× TE) was added and the mix was incubated at 50°C for an additional 2 h. The genomic DNA was then transferred to dialysis tubing with a 1000-kD molecular weight cutoff (Spectrum Laboratories) and dialyzed overnight at room temperature in 0.5× TE buffer. Dialyzed DNA was transferred to a microcentrifuge tube and quantified using a Quant-iT Broad-Range dsDNA Assay Kit (Thermo Fisher Scientific). DNA was used directly following quantification.
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9

Mutational Analysis of Organoid-Derived DNA

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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).
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10

Quantifying gpt Mutation Frequency

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The gpt mutation assay was performed as described previously12 (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 previously12 (link),13 (link)).
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