DSB detection by PFGE was done as reported previously18 (link). Briefly, cells were cast into 0.8% agarose plug (2.5 x 105 cells/plug), digested in lysis buffer (100 mM EDTA, 1% sodium lauryl sarcosine, 0.2% sodium deoxycholate, 1 mg/ml proteinase-K) at 37 °C for 36–40 h, and washed in 10 mM Tris-HCl (pH 8.0)–100 mM EDTA. Electrophoresis was performed at 14 °C in 0.9% pulse field-certified agarose (Bio-Rad) using Tris-borate-EDTA buffer in a Bio-Rad Chef DR III apparatus (9 h, 120°, 5.5 V/cm, and 30- to 18-s switch time; 6 h, 117°, 4.5 V/cm, and 18- to 9-s switch time; and 6 h, 112°, 4 V/cm, and 9- to 5-s switch time). The gel was stained with ethidium bromide and imaged on Uvidoc-HD2 Imager. Quantification of DSB was carried out using ImageJ software64. Relative DSB levels were calculated by comparing the results in the treatment conditions to that of the DSB level observed in untreated controls.
Pulse field certified agarose
Manufactured by Bio-Rad
Pulse Field Certified Agarose is a specialized agarose designed for use in pulsed-field gel electrophoresis (PFGE) applications. It is optimized to provide consistent and reliable separation of large DNA molecules. The agarose is certified for use in PFGE techniques.
Automatically generated - may contain errors
7 protocols using pulse field certified agarose
1
Double-Strand Break Detection by PFGE
2
Pulsed-field Gel Electrophoresis for DNA Damage
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Sub-confluent cultures of U2OS were treated with vehicle alone (DMSO), camptothecin (CPT 1 μM), or Ru65 (50 μM) and were either non-irradiated or UV-A irradiated. Cells were harvested by trypsinization, and agarose plugs of 106 cells were prepared in a disposable plug mold (Bio-Rad). Plugs were incubated in lysis buffer (100 mM EDTA, 1% (w/v) sodium lauryl sarcosyl, 0.2% (w/v) sodium deoxycholate, 1 mg ml–1 proteinase K) at 37 °C for 72 h, and washed four times in 20 mM Tris–HCl pH 8.0, 50 mM EDTA before loading onto an agarose gel. Electrophoresis was performed for 23 h at 14 °C in 0.9% (w/v) Pulse Field Certified Agarose (Bio-Rad) containing Tris-borate/EDTA buffer according to the conditions described in47 (link) and adapted to the Bio-Rad CHEF DR III apparatus. The gel was finally stained with ethidium bromide (EtBr) and analyzed using an Alpha Innotech Imaging system.
3
Pulsed-Field Gel Electrophoresis for DNA Double-Strand Break Analysis
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ES cells were transfected or treated as indicated and cells were harvested by trypsinization. Agarose plugs of 2.5 × 105 cells were prepared in a disposable plug mold (Bio-Rad Laboratories). Plugs were then incubated in lysis buffer (100 mM EDTA, 1% (wt/vol) sodium lauroyl sarcosinate, 0.2% (wt/vol) sodium deoxycholate, and 1 mg ml−1 proteinase K) at 37 °C for 72 h. Plugs were then washed four times in 20 mM Tris-HCl, pH 8.0 and 50 mM EDTA before loading onto an agarose gel. Electrophoresis was performed for 21 h at 14 °C in 0.9% (wt/vol) Pulse Field Certified Agarose (Bio-Rad Laboratories) containing Tris-borate/EDTA buffer in a pulse field gel electrophoresis (PFGE) apparatus (CHEF DR III; Bio-Rad Laboratories), according to the following protocol (block I: 9 h, 120° included angle, 5.5 V cm−1, 30–18 s switch; block II: 6 h, 117° included angle, 4.5 V cm−1, 18–9-s switch; block III: 6 h, 112° included angle, 4.0 V cm−1, 9–5 s switch). The gel was then stained with ethidium bromide and analysed by the AlphaImager system (ProteinSimple). Relative DNA DSB) levels were assessed by comparing DSB signals for each treatment to the background levels observed in untreated conditions using ImageJ.
4
Genetic Profiling of Listeria monocytogenes
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The genetic profile of L. monocytogenes isolates was subjected to DNA profiling using the restriction enzymes AscI and ApaI (Thermo Fisher Scientific, USA). It was then identified by electrophoresis using the CH-DR II (Bio-Rad Inc, Hercules, CA, USA) system according to the PFGE PulseNet protocol (CDC 2017). After the digestion of DNA in the prepared plugs, they were subjected to electrophoresis in 1% (w/v) Pulse Field Certified Agarose (BioRad). The values of the electrophoretic parameters used were: initial running time, 4.0 s; final running time, 40.0 s; total time 22 h; angle, 120°; 6.0 V/cm; temperature, 14°C; ramp factor, linear. The PFGE gels were stained with EZ-Vision (Amresco Inc., USA) for 30 minutes after electrophoresis. They were then destained with deionized water twice for 15 min. Phylogenetic dendrograms were drawn using BioNumerics 7.6 (Applied Maths, Belgium) with the Unweighted Pair Group Method and Arithmetic Mean (UPGMA) method (Dice similarity coefficient, 1.5% band tolerance, 0.5% optimization and 80% degeneracy cutoff value).
5
Linearization of BACs via PFGE
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The successful linearization of the BACs has been assessed through PFGE using the Clamped Homogeneous Electric Fields technology. Two percent low-melting agarose solution (CertifiedTM Low Melt Agarose, BioRad) was prepared using 1× TBE buffer (Tris base 1 M, Boric Acid 1 M, EDTA 0.02 M) and melted using a microwave. The solution was equilibrated at 50 °C in a water bath. One microgram of linearized BAC DNA was combined with equal volume of 2% CertifiedTM Low Melt Agarose and mixed by pipetting, to achieve a final concentration of 1% agarose. The mixture was quickly transferred to plug molds and left to solidify for 10 min. While waiting for the solidification of the plugs, 1% agarose gel (Pulse Field Certified Agarose, BioRad) was prepared using 1× TBE buffer and casted in a PFGE casting stand. Finally, the plugs were inserted inside the wells. PFGE was run according to the following program for 20 h: switch time 200 s, reorientation angle 120 °C, voltage gradient 5 V/cm. The final result was visualized through a transilluminator by staining the gel with EtBr for 1 h in 1× TBE.
6
Double-Strand Break Detection by PFGE
7
Linearization Assessment of BACs via PFGE
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The successful linearization of the BACs has been assessed through Pulse Field Gel Electrophoresis (PFGE) using the Clamped Homogeneous Electric Fields (CHEF) technology. 2% low melting agarose solution (Certified TM Low Melt Agarose, BioRad) was prepared using 1X TBE buffer (Tris base 1M, Boric Acid 1M, EDTA 0,02M) and melted using a microwave. The solution was equilibrated at 50°C in a water bath. 1 µg of linearized BAC DNA was combined with equal volume of 2% Certified TM Low Melt Agarose and mixed by pipetting in order to achieve a final concentration of 1% agarose. The mixture was quickly transferred to plug molds and left to solidify for 10 minutes. While waiting for the solidification of the plugs, 1% agarose gel (Pulse Field Certified Agarose, BioRad) was prepared using 1X TBE buffer and casted in a PFGE casting stand. Finally, the plugs were inserted inside the wells. PFGE was run according to the following program for 20 hours: The analysis was conducted with two separate biological replicates. The conditions used for the qPCR were 95°C 1 min initial denaturation, 95°C 30 s, 65°C 30 s, 72°C 30 s for 40 cycles with fluorescence detection after every elongation step. The PCR products were not longer than 250 bp and contained a GC content of ∼50%. The experiment was performed on an Eppendorf Realplex 2 Mastercycler.
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