Genomic DNA Extraction and Fragmentation

Genomic DNA was isolated from a 5-day-old culture of alphaΔssgB^{7 (link)} using phenol:chloroform extraction¹⁰. Briefly, the cell pellet was resuspended in 10.3% (w/v) sucrose containing 0.01 M ethylenediamine tetraacetic acid (EDTA, 20296.291, VWR Chemicals BDH) pH = 8, following lysis with 10% (w/v) sodium dodecyl sulfate (SDS, 20765.02, Serva). Extraction with phenol:chloroform (1:1 mix of phenol, 10001173, Fisher BioReagents^tm and chloroform, 32211, Honeywell) was performed and the nucleic acids were precipitated using isopropanol (33539, Honeywell). The pellet was dissolved in Tris-EDTA buffer (Trizma® base, RDD008, Sigma-Aldrich), followed by RNase A (EN0531, Thermo Fisher) and Proteinase K treatment (19131, Qiagen). The gDNA was isolated using phenol:chloroform extraction and precipitated using absolute ethanol (5250501, Biosolve) before resuspension in nuclease-free water. Fragmented gDNA was obtained by beat-beating the intact gDNA for 12 min using 2 mm diameter glass beads in a Mikro-Dismembrator U (Sartorius) at 2000 rpm. Chromosomal DNA concentrations were verified using the Quant-IT™ Broad-Range dsDNA Assay Kit (Q33130, Invitrogen).

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Kapteijn R., Shitut S., Aschmann D., Zhang L., de Beer M., Daviran D., Roverts R., Akiva A., van Wezel G.P., Kros A, & Claessen D. (2022). Endocytosis-like DNA uptake by cell wall-deficient bacteria. Nature Communications, 13, 5524.

Publication 2022

chloroform isopropanol Trizma® base RNase A Proteinase K Mikro-Dismembrator U Quant-IT™ Broad-Range dsDNA Assay Kit

Absolute ethanol Assay Cell Chloroform Chromosomal Dsdna Edta Genomic Isopropanol Nucleic acids Phenol Proteinase k Rnase Sodium dodecyl sulfate Sucrose Tris buffer Trizma

Corresponding Organization :

Other organizations : Leiden University, Radboud University Nijmegen, Radboud University Medical Center

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Variable analysis

independent variables

Fragmentation of genomic DNA by beat-beating for 12 minutes using 2 mm diameter glass beads in a Mikro-Dismembrator U at 2000 rpm

dependent variables

Chromosomal DNA concentrations

control variables

Cell culture age (5-day-old)
Strain used (αΔssgB7)
Lysis with 10% (w/v) sodium dodecyl sulfate (SDS)
Phenol:chloroform extraction
Isopropanol precipitation
RNase A treatment
Proteinase K treatment
Ethanol precipitation

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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