Genomic DNA Digestion and Southern Blot Analysis

Genomic DNA was prepared from 1x10⁸ cells according to standard protocols and digested with ApaI/XhoI, ApaI/NcoI and ApaI/SacII (New England Biolabs) for analysis of CEB1-1.8 I, CEB1-1.8 II and CEB1Gmut-1.7, respectively [31 (link), 63 (link)]. The digested DNA was resolved in 1% agarose gel and blotted onto Hybond-XL membrane (GE Healthcare). After transfer, the membrane was cross-linked with UV and hybridized with CEB1-0.6 and CEB1 Gmut probes for CEB1-1.8 and CEB1Gmut -1.7, respectively. ³²P labelling of DNA probes was performed by random priming using Klenow fragment exonuclease (New England Biolabs), in presence of [α-³²P]-CTP and hybridizations were performed in Church buffer at 55°C. Radioactive signals were detected using a BIORAD molecular imager FX.

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Maestroni L., Audry J., Luciano P., Coulon S., Géli V, & Corda Y. (2020). RPA and Pif1 cooperate to remove G-rich structures at both leading and lagging strand. Cell Stress, 4(3), 48-63.

Publication 2020

ApaI/SacII Hybond-XL membrane Klenow fragment exonuclease

Agarose Apai Buffer Ceb1 Cells Dna probes Exonuclease Genomic Hybridizations Klenow fragment Membrane Radioactive

Corresponding Organization :

Other organizations : Institut Paoli-Calmettes, Centre National de la Recherche Scientifique, La Ligue Contre le Cancer, Inserm, Centre de Recherche en Cancérologie de Marseille

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Variable analysis

independent variables

Restriction enzymes used for DNA digestion: ApaI/XhoI, ApaI/NcoI, and ApaI/SacII

dependent variables

DNA fragment sizes resulting from the restriction enzyme digestions, as observed by agarose gel electrophoresis and Southern blotting

control variables

Cell number (1x10^8 cells) used for genomic DNA preparation
Standard protocols used for genomic DNA preparation
Agarose gel concentration (1%) used for DNA fragment separation
Hybond-XL membrane used for DNA transfer
UV cross-linking of membrane after transfer
Probes used for hybridization: CEB1-0.6 and CEB1 Gmut
Hybridization conditions (Church buffer at 55°C)
Radioactive detection method (BIORAD molecular imager FX)

positive controls

None explicitly mentioned

negative controls

None explicitly mentioned

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