Advanced Search
Comet Assay for DNA Damage
The comet assay was performed using the CometAssay Kit (Trevigen) with minor modifications.
(1) Cracking. Place a petri dish containing 1 mL of ice-cold solution (1ⅹ phosphate-buffered saline and 20 mM EDTA). Chop 0.2 g of anther sample in the petri dish with a scalpel. A 100-mm nylon mesh filter (Millipore) was used to filter the nuclei mixed.
(2) Sample loading. 50 μL of the nuclei mixed into 300 μL of 1% (w/v) low-melting agarose (LMA agarose, pre-incubated at 37°C) and pipetted onto slides. After coagulation, immerse the slides in 4°C lysis solution (kit provided) for 1 h. Pay attention to handle all process gently.
Note: the LMA agarose should be melted at 90–100°C water bath for 5 minutes and placed in a 37°C water bath for at least 20 minutes to cool before use.
(3) Unspin. Drain excess buffer from slides and immerse in freshly prepared Alkaline Unwinding Solution (0.3 N NaOH, 5 mM EDTA) for 40 minutes at 4°C, in the dark. Then wash the slides with 1ⅹ TBE for 5min and repeat 2 times.
(4) Electrophoresis. Take out the slides and slides were electrophoresed at 25 V in 1ⅹ TBE for 30 min and then immersed in 70% (v/v) ethanol for 5 min.
(5) Dyeing and take pictures. After drying at room temperature, the slides were stained with SYBR green (1:10,000 dilution; Bio-Rad) and then examined with a Zeiss ImagerA2 microscope. The CometScore software (http://www.autocomet.com) was used to evaluate the percentage of DNA in each comet tail. Use CometScore software (http://www.autocomet.com) to evaluate the percentage of DNA (T DNA%) in each comet tail. The degree of DNA damage of different samples was calculated according to the method described by Wang and Liu (2006).
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Share
Bluesky
X
Copy link