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1. | Equipment and non-chemical materials | ||||
1.1 | Electrophoresis power supply: Thermo EC 1000-90 or alternative | ||||
1.2 | Electrophoresis tank: Large Fisher Scientific Recirculating Horizontal System or alternative | ||||
1.3 | Frosted slides: Surgipath or alternative | ||||
1.4 | Coverglasses: Surgipath 24 x 50 mm or alternative | ||||
1.5 | Microcentrifuge and microcentrifuge tubes | ||||
1.6 | Various micro-pipet and pipet tips | ||||
1.7 | Disposable test tubes | ||||
1.8 | Dishes for lysis of cells: Wheaton 900170 | ||||
1.9 | Nikon E400 Fluorescence microscope or equivalent | ||||
1.10 | Refrigerator and other general laboratory items, e.g. balance and pH meter | ||||
2. | Chemicals and reagents | ||||
2.1 | 10 x concentrated Dulbecco's PBS, Mg2+ and Ca2+ free | ||||
NaCl (Sigma S7653) | 80 g | ||||
KCl (Fisher Scientific P217-500) | 2 g | ||||
Na2HPO4.2H2O (Fisher Scientific S472-500) | 11.5 g | ||||
KH2PO4 (Sigma P0662) | 2 g | ||||
Adjust pH to 7.2 and q.s. to 1 liter with dd H2O, autoclave store at RT. | |||||
2.2 | Dulbecco's PBS, Mg2+ and Ca2+ free | ||||
Dilute 10 times of solution 3.1 with dd H2O, final pH 7.3 ~ 7.4. Autoclave at 121°C for 20 min, store at RT. | |||||
2.3 | Lysing solution for the comet assay (equivalent to 2 liters in the final preparation with the addition of 1% Triton X-100 and 10% DMSO) | ||||
2.4 g Tris (Sigma T6066), 74.4 g Na2EDTA.2H2O (Sigma E5134), 292.2 g NaCl (Sigma S7653), appropriate amount of H2O. | |||||
Set pH to 10 with NaOH pellets (Sigma S0899), q.s. to 1780 ml with dd H2O, store at RT. | |||||
2.4 | 10 N NaOH (Sigma S0899) stock solution (Caustic!!! Handle with care) | ||||
200 g q. s. to 500 ml with dd H2O, store at RT. | |||||
2.5 | 200 mM EDTA (Sigma E5134) stock solution | ||||
Dissolve 74.4 g in dd H2O, set pH to 10 with NaOH, q. s. to 1000 ml with dd H2O, store at RT. | |||||
2.6 | 0.5% low melting point agarose (LMPA) | ||||
0.5 g + 100 ml PBS(A), boil in microwave oven to dissolve agarose, compensate the evaporated H2O, aliquot the agarose into 15 ml tubes (~3 ml/ tube) before gelation and store the tubes under 4°C. | |||||
2.7 | 1.0% normal melting point agarose (NMPA) | ||||
1 g + 100 ml dd H2O, microwave to dissolve agarose, compensate the evaporated H2O, cool down to around 60°C before use. | |||||
2.8 | Neutralizing buffer - 0.4 M Tris (Sigma T6066) | ||||
Dissolve 48.5 g in dd H2O, set pH 7.4 with HCl and q. s. to 1000 ml with dd H2O, store at RT. | |||||
2.9 | Electrophoresis solution 300 mM NaOH, 1 mM EDTA | ||||
Mix 48 ml of 10 N NaOH, 8 ml of 200 mM EDTA and 1544 ml dd H2O. Chill to 4°C before use. | |||||
2.10 | Fluorescent staining solution 250 x Strength Stock Ethidium bromide (Sigma E8751, DNA poison!!! Handle with care) in dd H2O 5 mg/ml, store at RT. |
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2.11 | Staining solution | ||||
Dilute solution 3.10 250 time to 20 µg/ml with dd H2O, store at RT. | |||||
2.12 | Dimethyl sulfoxide (Fisher Scientific D128-500) | ||||
2.13 | Triton X-100 (Sigma T9284) | ||||
3. | Operation procedures | ||||
3.1 | Material and reagent preparation | ||||
3.1.1 | Mix 89 ml lysing solution (3.3) with 1 ml of Triton X-100 and 10 ml of DMSO in a staining dish, chill the mix to 4°C before use. | ||||
3.1.2 | Microwave the LMPA and NMPA aliquots to complete molten state (in a container filled with water), check and shake the tube every a few seconds to avoid overheat and explosion. Leave the molten LMPA in 40°C bath and NMPA in 45°C for at least 10 min before use. | ||||
3.2 | Slide preparation | ||||
Note: Steps 3.2.1 may be carried out days before Comet assay experiment. | |||||
3.2.1 | Dip ¾ of clean frosted slides (unfrosted portion), one at a time, in molten NMPA. Scrape agarose off the underside. Leave the slides horizontally to dry. A slide warmer may be used to facilitate the drying of slides. | ||||
Note: Conduct the steps 3.2.2 to 3.2.3 under dimmed light to prevent the occurrence of DNA damage. | |||||
3.2.2 | Mix about 10,000 cells with 75 µl of molten 0.5% LMA at 37°C. Pipette the cell suspension onto the first agarose layer, and spread the mixture with a coverglass, and maintain the slide on an ice-cold flat tray for 30 min. | ||||
3.2.3 | After removal of the coverglass, add the third layer of 0.5% LMA (70 µl) at 37°C, spread gel using a coverglass and again allow the gel to solidify on ice for 10 min. | ||||
3.2.4 | After removal of the coverglass, immerse the slide in freshly prepared cold lysing solution for a minimum of 1 h at 4°C. | ||||
3.3 | Electrophoresis | ||||
3.3.1 | Remove the slides from the lysing solution, drain the slides and rinse briefly with cold dd H2O. Place in a horizontal gel electrophoresis tank side by side, avoiding spaces and nearest to the anode. Fill the electrophoresis tank with 1.6 L of cold electrophoresis solution. | ||||
3.3.2 | Leave the slides in the solution for 30 min to allow the unwinding of the DNA and expression of alkali-labile damage. | ||||
3.3.3 | Perform electrophoresis at 4°C for 30 min (or other desirable time) at a constant voltage (0.8 V/cm). | ||||
3.4 | Staining | ||||
3.4.1 | After electrophoresis, Place the slides horizontally and gently add Tris buffer (0.4 M Tris, pH 7.5) dropwise to neutralize the excess alkali. Let the slides sit for 5 min. This neutralizing procedure was repeated three times. | ||||
3.4.2 | Add 60 µl EtBr (20 µg/ml) to each slide and cover it with a coverglass. Let the slides sit for 5 min at the horizontal position. Place the slides in a humidified airtight container to prevent drying of the gel. Analyze the slides within 3-4 h. | ||||
3.5 | Slide scoring Randomly score the comet-like nuclei using the Comet Assay Image Analysis System. For each treatment score a minimum of 50 cells or 25 cells per slide is required. |