Pulsed-field Gel Electrophoresis Protocol

PFGE was performed as previously described (Beck et al, 2010 (link)). Briefly cells were embedded in a 0.8% agarose plugs (2.5x10⁵ cells/plug), digested in lysis buffer [100mM EDTA, 1%(w/v) sodioum lauryl sarcosyne, 0.2% (w/v) sodium deoxycholate and 1mg/ml proteinase K] at 37°C for 48h and washed in 10mM Tris-HCl pH8.0 and 100mM EDTA. Electrophoresis was performed at 15°C in 0.8% (w/v) PFGE certified agarose (Bio-Rad Laboratories) with Tris-borate/EDTA buffer employing a CHEF DR III apparatus (9h, 120°), 5.5V/cm, 30-18s switch time)(Bio-Rad). The gel was stained with ethidium bromide.

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Galanos P., Vougas K., Walter D., Polyzos A., Maya-Mendoza A., Haagensen E.J., Kokkalis A., Roumelioti F.M., Gagos S., Tzetis M., Canovas B., Igea A., Kanavakis E., Kletsas D., Roninson I., Garbis S.D., Nebreda A., Thanos D., Townsend P., Blow J.J., Sørensen C.S., Bartek J, & Gorgoulis V.G. (2016). Protracted p53-independent stimulation of p21WAF1/Cip1 fuels genomic instability by deregulating the replication licensing machinery. Nature cell biology, 18(7), 777-789.

Publication 2016

PFGE certified agarose CHEF DR III apparatus

Agarose Buffer Cells Edta Electrophoresis Ethidium bromide Pfge Proteinase k Sodium deoxycholate Tris Tris borate edta buffer

Corresponding Organization : Palacký University Olomouc

Other organizations : National and Kapodistrian University of Athens, Academy of Athens, Biomedical Research Foundation of the Academy of Athens, University of Copenhagen, Danish Cancer Society, University of Dundee, Institute for Research in Biomedicine, University of Zurich, National Centre of Scientific Research "Demokritos", University of South Carolina Sumter, University of Southampton, Manchester Academic Health Science Centre, University of Manchester

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Variable analysis

independent variables

Agarose concentration (0.8%)
Lysis buffer composition [100mM EDTA, 1%(w/v) sodium lauryl sarcosyne, 0.2% (w/v) sodium deoxycholate and 1mg/ml proteinase K]
Lysis buffer incubation time (48h)
Electrophoresis conditions (15°C, 0.8% (w/v) PFGE certified agarose, Tris-borate/EDTA buffer, CHEF DR III apparatus, 9h, 120°, 5.5V/cm, 30-18s switch time)

dependent variables

DNA migration pattern (visualized by ethidium bromide staining)

control variables

Cell density (2.5x10^5 cells/plug)
Washing buffer (10mM Tris-HCl pH8.0 and 100mM EDTA)

controls

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

Annotations

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