Alkaline Single Cell Gel Electrophoresis: A Sensitive Technique for Quantitative Estimation of DNA Damage in Individual Cancer Cells Following Chemotherapy
Published: April 30, 2023
Abstract
Source: Lu, Y., et al. Evaluating In Vitro DNA Damage Using Comet Assay J. Vis. Exp. (2017)
In this video, we demonstrate an alkaline single-cell electrophoresis assay to quantify both single- and double-strand DNA breaks in doxorubicin-treated cells. Alkaline conditions disrupt the hydrogen bonds between DNA base pairs, resulting in the complete unwinding and separation of DNA strands. This allows differential migration of undamaged and damaged DNA fragments in agarose gel upon electrophoresis, revealing a comet-shaped structure of slowly migrating undamaged DNA forming the circular comet head, and rapidly migrating damaged DNA fragments forming the elongated comet tail.
Protocol
1. Prepare Reagents 1x PBS Dilute 100 mL 10x PBS with 900 mL dH2O and adjust the pH to 7.4 using a pH meter. Store at room temperature. Lysis solution (LS) Prepare 2.5 M NaCl, 100 mM disodium EDTA, 10 mM Tris base, and 200 mM NaOH in 900 mL dH2O; it commonly takes about 20 min to allow the mixture to fully dissolve. Adjust the pH to 10 using a pH meter. Add 1% sodium lauryl sarcosinate and …
Divulgazioni
The authors have nothing to disclose.
Materials
| 10x PBS(Ca++, Mg++ free) | TEKnova | P0196 | |
| NaCl | Sigma | S5886 | |
| EDTA | TEKnova | E0308 | |
| Trizma base | Sigma | T1503 | |
| NaOH | Sigma | 72068 | |
| Sodium lauryl sarcosinate | Sigma | L7414 | |
| Triton X-100 | Sigma | 93443 | |
| SYBR Green | Invitrogen | S33102 | |
| Low melting point agarose | Invitrogen | 16520 | |
| Agarose | Invitrogen | 16500 | |
| 95% ethanol | WARNER-GRAHAM | #64-17-5 | |
| Trypsin | GIBCO | 25300-054 | |
| Glass tissue slides | ELECTRON MICROSCOPY SCIENCES | 63422-11 | |
| Kimwipes | KIMberly-Clark | ||
| 1.5 mL Microcentrifuge Tubes | DENVILLE | ||
| Pipette Tips | SHARP | ||
| Microwave | Avanti | ||
| Waterbath | PRECISION | ||
| Horizontal electrophoresis chamber | TREVIGEN | Cometassay ES II | |
| Power supply | Bio-Rad | ||
| Incubator | Quincy Lab | Model 12-140E | |
| Fluorescent microscope | Zeiss | LSM700 | |
| Micropipettor | Eppendorf |
Citazione di questo articolo
Alkaline Single Cell Gel Electrophoresis: A Sensitive Technique for Quantitative Estimation of DNA Damage in Individual Cancer Cells Following Chemotherapy. J. Vis. Exp. (Pending Publication), e21114, doi: (2023).