The DNA substrates were prepared by amplifying a 5.7 kb plasmid using Pfu DNA polymerase (Promega, WI) and oligonucleotides carrying 5′-biotin (Midland, TX) in the presence of 32P-α-dATP (Perkin Elmer). PCR products were purified by gel-filtration with Sepharose CL-2B beads (Sigma-Aldrich, MO). Peak fractions were pooled and concentrated to 37.5 ng/μl. 5′ avidin DNA was prepared by pre-incubating 5′ biotin DNA (20 ng/μl) with Neutravidin (4 μg/μl) (Pierce/ThermoScientific, IL) for 10 minutes. A typical repair assay contained 5 μl non-depleted extracts, mock depleted extracts, or extracts depleted of Polθ, Ku70 or BRCA1. 0.5 μl 10x ATP mix (20 mM ATP/200 mM phosphocreatine/0.5 mg/ml creatine kinase/50 mM DTT), 1.5 ng/μl DNA, and ELB buffer (total volume = 7.5 μl). Reactions were incubated at room temp and samples taken at the indicated times were mixed with an equal volume of 2% SDS/25 mM EDTA. At the end, samples were brought up to 10 μl with H2O and treated with 1 μl proteinase K (10 mg/ml) at room temp for 2 hours. Products were separated on 1% TAE/agarose gels by electrophoresis and gels were dried and exposed to X-ray films. For analysis of repair junctions, the 2 kb fragment bordering the junction was amplified by PCR, subcloned into a pUC vector, and sequenced by the Sanger method (Genewiz, NJ).
Sepharose cl 2b beads
Manufactured by Merck Group
Sourced in Macao
Sepharose CL-2B beads are a type of agarose-based chromatography media used for the separation and purification of biomolecules. The beads have a controlled degree of cross-linking, which provides a stable and porous structure suitable for a variety of applications in biochemistry and molecular biology.
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3 protocols using sepharose cl 2b beads
1
DNA Repair Assay and Junction Analysis
2
Quantitative DNA Resection Assay
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The DNA substrates were prepared by amplifying a 5.7kb plasmid using Pfu DNA polymerase (Promega, WI, USA) and oligonucleotides carrying 5′-phosphotyrosine, biotin or hydroxyl groups (Midland, TX, USA) in the presence of 32P-labeled dATP. The products were purified first by Qiagen's polymerase chain reaction (PCR) purification columns and then by gel-filtration with Sepharose CL-2B beads (Sigma-Aldrich, MO, USA). The peak fractions were pooled and concentrated to 50 ng/μl. The 5′ avidin DNA was prepared by pre-incubating at 20 ng/μl of 5′ biotin DNA with 4 μg/μl Neutravidin (Pierce/ThermoScientific, IL, USA) for 10 min. A typical resection assay contained 5 μl depleted cytosol, 0.5 μl 10x ATP mix (20 mM ATP/200 mM phosphocreatine/0.5 mg/ml creatine kinase/50 mM DTT), 1–1.5 ng/μl DNA, and ELB buffer or DNA2 protein (total volume = 7.5 μl). The reactions were incubated at 22ºC and samples were taken at the indicated times and mixed with an equal volume of 2% SDS/25 mM EDTA. At the end, samples were brought up to 10 μl with H2O and treated with 1 μl proteinase K (10 mg/ml) at 22ºC for 2 h. The resection products were separated by 1% TAE/agarose gel electrophoresis and the gels were dried and exposed to Phosphorimager (Fuji) and film.
3
DNA Repair Assay and Junction Analysis
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