Recently we’ve started to set up comet assay in our lab. As the first experience we are face with some problems and questions. We are so grateful to help us in this attempt.
Our questions are as follows:
1- According to the time consuming process, which steps can we pause the test to continue in the next days? And what are storage conditions in these periods?
2- Our LMA does not melt at 37 °C and even near temperatures!? Is there a particular reason?
Dear Nahid Ahmadi,
you can make all working solutions and prepare slides with agarose in one day, next day, you should perform such steps as lysis and electrophoresis,and then you can perform the staining and analysis. What type of LMA do you use? You should use the type IV.
Thank you very much for your attention
Actually we followed the instruction you mentioned. We'll be so pleased to know if we can fulfill the step 2 (lysis and electrophoresis) in multiple days.
According to the attached product information sheet of agarose, do you suggest us to use agarose type IV(A3643 SIGMA) instead of LMA (A9414 SIGMA)?
Sincerely
Nahid
I agree with Maxim too, and I would add that you can perform the lysis step overnight at 4 degrees (protected from light) to continue the next day with the electrophoresis. I used to do that and results were not affected. So if you don't have enough time you can do the whole procedure in 3-4 different days. Regarding the agarose, I used this one from Bio-Rad and it worked perfect (Certified Low-Melt Agarose #161-3111). Hope that helps!
Dear Rodrigo Guzman
Thank you so much for your attention and helpful answers.
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