Hi everyone! I am looking for an efficient comey assay protocol in chlamydomonas reinhardtii if you have some suggestions let me know thanks a lot!
Hola, en nuestro laboratorio se hizo el ensayo de cometa con Chlamydomonas reinhardtii expuesta a diferentes contaminantes en varias ocasiones.
Publicación: Prado, R., Garcia, R., Rioboo, C., Herrero, C., Abalde, J., Cid, a., 2009. Comparison of the sensitivity of different toxicity test endpoints in a microalga exposed to the herbicide paraquat. Environ. Int. 35, 240–247. https://doi.org/10.1016/j.envint.2008.06.012.
Publicación: Esperanza, M., Cid, Á., Herrero, C., Rioboo, C., 2015. Acute effects of a prooxidant herbicide on the microalga Chlamydomonas reinhardtii: Screening cytotoxicity and genotoxicity endpoints. Aquat. Toxicol. 165, 210–221. https://doi.org/10.1016/j.aquatox.2015.06.004.
The comet assay protocol used is a modification ofthe original protocol (Singh et al., 1988) adapted to planktonic algae by Erbes et al. (1997), with an additional modification that is the DNA staining by adding 50 µl SYBR Green (SYBR Green I Nucleic acid Gel Stain) 100× to each slide, and not the conventional DAPI or ethidium bromide staining.
Espero que ta valga.
Un saludo
Dear Margarita Toledo,
Chlamydomonas made with only a single cell. So, my suggestion is, better you try with the method of Cabrita et al., 2005. Because of its the simplest method for analyzing the DNA integrity in a single cell organism, especially for sperm cells. To analysis the DNA integrity (Comet assay), you should take a minimum of 100 to 200 animals for a single test. usually, maximum cell count needs to authenticate the fragmentations of tail DNA.
I have two ideas for the isolation of Cell from Chlamydomonas
1. Just homogenize the 100 to 200 organisms and treated with lysis (digestion) buffer.
then proceed with the usual protocol of Cabrita et al. (2005) or Singh et al. (1988).
2. Directly treat the organisms with lysis (digestion) buffer and proceed with the usual protocol of Cabrita et al. (2005) or Singh et al. (1988).
Finally, DNA integrity will be analyzed with the help of CASP software, which is freely available.
I think it may give some ideas for your studies.
With regards
S.Thirunavukkarasu
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