After treatment with compounds, DNA was automatically purified using the Agencourt DNAdvance Genomic DNA Isolation Kit and an automatic dispenser (Biomek i5 or NXp, Beckman Coulter) according to the manufacturer’s instructions. Briefly, the cells were washed 3 times with PBS and incubated with 200 μl of Lysis buffer containing 50 mM dithiothreitol (DTT) and proteinase K at 55 °C for 1 h with constant agitation. The lysates were transferred to 96-well 1.2-ml deep well storage plates (Thermo, AB1127) and mixed with 100 μl of Bind1 buffer, and then the 170 μl of Bind2 buffer was then added, and the samples were again mixed. After collection of the DNA-binding magnetic beads, the beads were washed twice with 340 μl of 70% ethanol. DNA was eluted with 200 μl of MilliQ water.
Dnadvance genomic dna isolation kit
Manufactured by Beckman Coulter
The DNAdvance Genomic DNA Isolation kit is a product designed for the extraction and purification of genomic DNA from a variety of sample types. It utilizes a simple and efficient silica-based method to isolate high-quality DNA suitable for downstream applications such as PCR, sequencing, and other molecular biology techniques.
Automatically generated - may contain errors
Lab products found in correlation
Solution se, by Lonza (2 mentions)
24 well plate, by Corning (2 mentions)
Ab1127, by Thermo Fisher Scientific (1 mentions)
Biomek i5, by Beckman Coulter (1 mentions)
Biomek nxp, by Beckman Coulter (1 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Nippon Gene (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Infinium global screening array 24 v2.0 microarray, by Illumina (1 mentions)
8 protocols using dnadvance genomic dna isolation kit
1
Automated DNA Extraction from Cells
2
DNA and RNA Purification from HepG2 Cells
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Total DNA was purified from REP-HepG2-NTCP cells and naive HepG2-NTCP-C4 cells using an Agencourt DNAdvance Genomic DNA Isolation kit and a Biomek NXp automatic dispenser from Beckman Coulter according to the manufacturer’s instructions. Total RNA was purified using an Agencourt RNAdvance Cell v2 and a Biomek NXp automatic from Beckman Coulter according to the manufacturer’s instructions. Before elution, the RNA samples were treated with five units DNase I (Nippon Gene) at 25°C for 15 min. For the microarray analysis, total RNA was purified from HepG2 cells using a miRNeasy Mini kit (Qiagen) according to the manufacturer’s recommendations, and samples were treated with 1 mL of DNase at 37°C for 30 min using a TURBO DNA-free kit (Ambion). DNA and RNA sample concentrations were determined using a DeNovix spectrophotometer.
3
Genetic Sex Identification in Fish
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At 10 months of age (September 2016) all fish were placed in a common garden setup; fshr crispants displaying partial or full loss of pigmentation (n = 137) together with an equal number of noninjected control (pigmented) fish. At 16 months of age, all fish were fin clipped and passive integrated transponder tagged for identification. Genomic DNA (gDNA) was extracted from the sampled fin tissue using the DNAdvance Genomic DNA Isolation kit (Beckman Coulter, Inc.) according to the manufacturer’s protocol. We also identified the genetic sex by assaying the sdy alleles on gDNA according to Ayllon et al (26 (link)) but with the following corrected primers and probe: Ss_sdY_Exon2_F: 5′-CCTACAAGCCCTTCTCCCTGAT-3′; Ss_sdY_Exon2_R: 5′-GGGCTTTGGGAGAGAGATGAC-3′; Ss_sdY_Exon2_Pro: 5′-VIC-ATGGATGGGATCCC-MGBNFQ-3′; Ss_sdY_Exon4_R: 5′- GGAGGACTCAAGCCAGATCCT-3′.
4
Targeted CRISPR genomic DNA isolation
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Genomic DNA was extracted from cells using the Agencourt DNAdvance genomic DNA isolation kit (Beckman Coulter Agencourt DNAdvance—Nucleic Acid Isolation from Mammalian Tissue #A48706) according to the manufacturer's directions. The CRISPR targeted genomic region of HBB was PCR amplified for subsequent Sanger sequencing using primers F1_HBB and R_HBB. Amplification was performed in a 50 μl reaction volume, consisting of 10 μl of 5 × Phusion HF buffer, 0.5 μM forward primer, 0.5 μM reverse primer, 200 μM dNTP, 1.5 μl DMSO, 0.5 μl of Phusion polymerase and 100 ng of gDNA template. PCR conditions were as follows: 30 s at 98 °C for initial denaturation, followed by 30 cycles of 10 s at 98 °C for denaturation, 15 s at 64 °C for annealing, 30 s at 72 °C for extension and 5 min at 72 °C for the final extension. For Supplementary Fig. 2f , we used primers HBB_primer_1_3F and HBB_primer_1_3R to amplify a longer region of the HBB locus for subsequent Sanger sequencing. The conditions were as above with 62 °C for annealing. To amplify the HBD locus to assay for off-target activity primers HBD_primer_F and HBD_primer_R were used with 64 °C for annealing. For primer sequences used see Supplementary Table 3 .
5
Estimating Pairwise Relatedness using DNA Genotyping
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To estimate pairwise relatedness among individuals in the sample, DNA was collected using the Oragene-Discover saliva kit (DNA Genotek; OGR-500). DNA was extracted using the DNAdvance genomic DNA isolation kit (Agencourt; A48705), and individuals were genotyped on the Infinium Global Screening Array-24 v2.0 microarray (Illumina). Genotypes were called and quality filtered using default settings in GenomeStudio (32 ). We estimated pairwise relatedness using three sets of 2,500 randomly selected single nucleotide polymorphism (SNP) markers from the GSA microarray in the R package related (33 (link)). The estimated relatedness values were highly correlated across all three subsets (r > 0.90 and P < 0.001 for all pairwise correlations), and we therefore proceeded with the relatedness estimates calculated using one of the three subsets of markers for subsequent analyses.
6
Generating Cetuximab-Resistant Cell Lines
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The CCK-81 and NCI-H508 cell lines were incubated in a concentration range of ENU (0–10 mg/mL), and viability was measured after 48 h. A concentration of 0.1 mg/mL was subsequently selected for resistance models as having a modest effect on cell viability while still generating a high rate of mutations (Supplemental Fig. S11 ). Cells were incubated in ENU at the indicated concentration for 24 h before being washed three times with PBS and incubated in media for a further 24 h. Cells were then selected with 10 µg/mL Cetuximab 48 h post-ENU exposure for 8 wk. Clones were then picked using Scienceware small cloning cylinders and either transferred to 96-well plates or expanded into large flasks for drug sensitivity assays. DNA was extracted in 96-well plate format using the Agencourt DNAdvance Genomic DNA Isolation kit.
7
CRISPR-Cas9 Nucleofection in U2OS Cells
8
CRISPR-Cas9 Genome Editing in U2OS Cells
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One day before nucleofection, 80–90% confluent U2OS cells were passaged at a 1:2 dilution ratio into fresh media. Nucleofection was performed by pooling 3 × 105 U2OS cells per condition and spun down at 300g for 10 min, washed with 1× PBS, spun again, then resuspended in SE solution (10 µl per condition, Lonza). DNA mixtures were prepared by combining 750 ng of Cas9 plasmid, 250 ng of guide RNA plasmid, 5 pmol of the GUIDE-seq dsODN49 (link) and SE solution into a total volume of 12 µ. Each 10 µl aliquot of U2OS cells is combined with DNA mixture to a total volume of 22 µl, and nucleofected with program DN-100 on the 96-well Shuttle Device component of a 4D-Nucleofector system. Following nucleofection, cells were allowed to rest for 10 min before addition of 100 µl of prewarmed media per well. Each condition was then split into two 50 µl aliquots and plated on 24-well plates (Corning). Cells were cultured for 5 days postnucleofection, with media replacement after the first day. Following removal of media and a wash with 1× PBS buffer, genomic DNA was isolated using the DNAdvance Genomic DNA Isolation Kit (Agencourt), following the manufacturer’s protocols. Genomic DNA was stored at −20 °C until further use.
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