In our comet assays we use the below mentioned chemicals and solutions in our laboratory and our images are good to monitorize. We made slides by handmade. 

0.65 % low melting agarose

1% nomal melting agarose

cold lysing solution (2,5 M NaCl, 100 mM EDTA, 10 Mm Tris, 1% Triton X-100, 10% DMSO, pH: 10)

cold electrophoresis solution (10 N NaOH, 200 Mm EDTA, pH>13)

25 V, 300 mA electrophoresis

Neutralization buffer (0, 4 M Tris, pH: 7.5)

Ethidium bromid

Olympus BX51 fluorescant microscope

Comet Image Processing and Analysis Systems (BAB Bs 200 Pro) 

(In Deok Kim, Bae Jin Ha * Paeoniflorin protects RAW 264.7 macrophages from LPS-induced cytotoxicity and genotoxicity. Toxicology in Vitro 23 (2009) 1014–1019). The mentioned article as above will be helpful to you. 

Best regards 

Thank you very much, Fulya, for your kind reply. I checked the paper. Yes I'm sorry forgot to mention a fluorescent microscope is required for this, of course. we have it. but still if I may ask you to kindly clarify did you use specialized slide gel electrophoresis  unit for slides (such as this for example: https://www.trevigen.com/item/1/56/179/379/CometAssay_Electrophoresis_System_II/)  and whether you needed to have control cells of known damage extent (validated by another method for examlpe; so say you use cells that you know have around 50 breaks/nucleus to be able to say this is the expected tail length of X amount of damage) or is this not required at all (even at first when you start establishing your lab protocol for comet assay, don't you need to make sure that tails should look like this if they have roughly X breaks by using controls for standardization)

Thanks again so much for your help

we  dont use slides we just precoate slides by ourselves by LMA and NMA. we use H2O2 as a positive control. as a control slide non-treated cells were used. we measure tail lenght by programme. for each experiment we use positive and negative control. 

To your point about the elctrophoresis unit, we use a standard electrophoresis unit, however, we did purchase the specialized electrophoresis tank. 

The link below provides a nice protocol for preparing buffers/slides for the neutral comet assay:

http://www.nature.com/nprot/journal/v1/n1/full/nprot.2006.5.html

The alkaline comet assay utilizes an additional buffer.

We always prepare our own positive control, however, it is important to note that depending on what you are looking to see, e.g. double-strand breaks, interstrand crosslinks, etc. will require you to prepare your samples differently. H2O2 is a great positive control for looking at certain types of DNA damage, but it is not always a proper positive control. 

As an alternative for visualizing the DNA, we use SYBR Gold. 

@Fulya,

Thank you, I did a pilot experiment using the M&Ms in the paper you kindly listed. A few pics are attached, it didn't work well but I have many questions to ask that maybe when considered the assay can be more successful: 

1. How did you prepare your slides? dipping in NMPA didn't result in a leveled layer of agarose for me (any tips?) so I pipetted 1.5 ml of the molten agarose onto a slide, put parafilm and placed another slide on top, then removed the parafilm and top slide and this made a somewhat leveled layer... but yet some slight differences in width along the slide

2. Now when we add the PBS resuspended pelleted cells onto the LMPA at 37, agarose gelefies a little, what do you do ensure the cell+agarose mix stays liquid? how do you spread this volume into a leveled layer? and how much of the top 0.5%LMA do you use to make a final layer? (I think maybe i'm using too much agarose in all layers, resulting in poor staining in the end)

3.What concentration of H2o2 (%) do you use as positive control treatment and how long? i couldn't see any tails with 0.1% h2o2 for 20 mins

4. I feel the agarose is interfering with staining and seeing the signal, because no matter what focus/level I use, it looks just like when a nucleus is out of focus in regular ICC, i see a signal but i know it's not on this level..but i can't bring it to focus

5. Are tails easily recognizable at 20X? at which magnification would you visually see and identify a tail and head to the stained nucleus

I know it might sound straightforward but as a beginner I would extremely appreciate if someone can share their detailed process/tips for preparing the slides (the coating, the spreading of cells (how you do it) and the final coating and any insight onto why i got no tails with positive control

Thank you very much for reading