The CO-FISH assay was adapted from previous studies [35 (link), 36 (link)]. Briefly, exponentially growing cells were cultured overnight in the presence of 10 μM BrdU:BrdC (3:1) (Sigma-Aldrich) at 37 °C to allow for one round of replication. Colcemid (Roche) was then added at a concentration of 0.1 μg/ml for 4 h to arrest cells at prometaphase. After fixation of cellular preparations on slides and Hoechst 33258 (Sigma-Aldrich) staining, the newly synthesized strands were degraded following UV light exposure and treatment with 10 U/μl ExoIII (Promega). Hybridization was performed using fluorescent centromeric PNA probes against CENP-B box motif sequences. The PNA probe labeled with Cy3 (ATTCGTTGGAAACGGGA; PNABio Inc) hybridizes with the leading strand and the reverse PNA probe labeled with Alexa-488 (TCCCGTTTCCAACGAAT; Eurogentec) hybridizes with the lagging strand. DNA was counterstained with DAPI (Sigma-Aldrich). Metaphases were captured on a confocal laser microscope (FV1000 Olympus) and images were analyzed using Image J software (NIH). Quantitation to measure for SCE between α satellite sequences (C-SCE) was done by counting the number of CO-FISH signals showing C-SCE over the total number of CO-FISH signals observed for each metaphase.
Exoiii
Manufactured by Promega
ExoIII is a recombinant exonuclease enzyme that catalyzes the stepwise removal of mononucleotides from the 3' termini of DNA molecules.
Automatically generated - may contain errors
Lab products found in correlation
Axioimager m2 epiflourescence microscope, by Zeiss (2 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Colcemid, by Roche (1 mentions)
Brdu brdc, by Merck Group (1 mentions)
Alexa 488, by Eurogentec (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Fv1000, by Olympus (1 mentions)
Mnase, by New England Biolabs (1 mentions)
Dynabeads oligo dt 25, by Thermo Fisher Scientific (1 mentions)
Genomic tip 20 g column, by Qiagen (1 mentions)
Quick ligation kit, by New England Biolabs (1 mentions)
Klenow fragment, by New England Biolabs (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Agencourt ampure xp bead, by Beckman Coulter (1 mentions)
Fla 5000 phosphorimager, by Fujifilm (1 mentions)
5 protocols using exoiii
1
Chromosome-specific CO-FISH Assay
2
Telomere FISH and CO-FISH Analysis
3
RNA-seq and MNase-seq Library Preparation
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Total RNA was extracted as described above. Poly-A RNA was purified from 5 μg total RNA with Dynabeads Oligo (dT)25 (Thermo Fisher Scientific). Paired-end RNA libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England BioLabs). Sequencing of three biological replicates was performed using the Illumina HiSeq 2500 platform.
Chromatin was digested with 120 U of MNase (New England BioLabs) and 8 U of EXOIII (Promega), as previously described (47 (link)), and purified using the QIAGEN Genomic-tip 20/G column. Mononucleosome DNA was separated by agarose gel electrophoresis and purified using the Quantum Prep Freeze ‘N Squeeze DNA Gel Extraction Spin Column (Bio-Rad). Purified DNA was treated with NEBNext End Repair Module, followed by Agencourt AMPure XP beads (Beckman Coulter) cleanup, and dA (deoxyadenosine) tailing with Klenow fragment (New England BioLabs). After purification, the samples were ligated with Illumina TruSeq multiplexing primers using the Quick Ligation Kit (New England BioLabs) and amplified using the KAPA HiFi Ready Mix (Thermo Fisher Scientific). Following the final purification, the DNA was quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). Sequencing was performed on the Illumina HiSeq 2500 platform using three biological replicates.
Chromatin was digested with 120 U of MNase (New England BioLabs) and 8 U of EXOIII (Promega), as previously described (47 (link)), and purified using the QIAGEN Genomic-tip 20/G column. Mononucleosome DNA was separated by agarose gel electrophoresis and purified using the Quantum Prep Freeze ‘N Squeeze DNA Gel Extraction Spin Column (Bio-Rad). Purified DNA was treated with NEBNext End Repair Module, followed by Agencourt AMPure XP beads (Beckman Coulter) cleanup, and dA (deoxyadenosine) tailing with Klenow fragment (New England BioLabs). After purification, the samples were ligated with Illumina TruSeq multiplexing primers using the Quick Ligation Kit (New England BioLabs) and amplified using the KAPA HiFi Ready Mix (Thermo Fisher Scientific). Following the final purification, the DNA was quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). Sequencing was performed on the Illumina HiSeq 2500 platform using three biological replicates.
4
Telomere FISH and CO-FISH Analysis
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Telomeric FISH and CO-FISH were performed as previously described 43 (link). Cells collected during metaphase spread analysis were denatured and hybridized to a peptide nucleic acid (PNA) probe Alexa488-OO-(TTAGGG) at 37 C for 2 hours to label the telomeres. For CO-FISH, cells were cultured in the presence of 10 μM BrdU and BrdC (3:1) for one population doubling before colcemid treatment. Slides containing metaphase spreads were incubated with Hoechst 33258 for 15 min, exposed to UV for 30 min, treated with ExoIII (Promega) for 10 min, and then hybridized to the PNA Alexa488-OO-(TTAGGG) and Cy3-OO-(CCCTAA)3 probes at room temperature for 2 hours. DNA was counterstained with DAPI. Slides were then washed, dehydrated by washing in an ethanol series, and imaged using a Zeiss AxioImager M2 epiflourescence microscope.
5
Exonuclease III Footprinting of hrpJ-hrpL Promoter
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Exonuclease III (ExoIII) footprinting was performed on variant PhrpJ-hrpL double-stranded DNA (dsDNA) promoter probes, labeled at the PhrpJ terminus with a 5′ cyanine (Cy3) dye molecule during PCR amplification using pTE103-PhrpJ and pTE103-PhrpJ(Δ35e) as the templates. Footprinting reactions (using 12-μl reaction mixtures) were performed in STA buffer (2.5 mM Tris-acetate [pH 8], 8 mM Mg-acetate, 10 mM KCl, 1 mM dithiothreitol, 3.5% [wt/vol] polyethylene glycol [PEG] 8000). The copurification of RNAP-HrpL and RNAP-HrpLΔR4.2 complexes is described in Text S1 . IHF and σ54 protein samples were sourced from the laboratory collection and purified as previously described (15 (link)). Protein-DNA complexes were preincubated at 22°C for 10 min before the addition of 50 units ExoIII and proprietary buffer (Promega). Digestion was performed for 2 min before ExoIII was inactivated with 20 mM EDTA for 10 min at 70°C. Partially digested single-stranded DNA (ssDNA) products were run on an 8% urea footprinting gel. Cy3 fluorescence was detected using a FLA-5000 phosphorimager (Fujifilm) with a 488-nm excitation laser and a 532-nm emission filter.
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