Hi, I'm doing the Alkaline COMET assay. But since I have many samples I wanted to know if it is possible to preserve my slides after doing the electrophoresis?
I want to preserve the slides, so that I can stain and score them at a later date.
Jhoti
Neutralizing in tris, washing in water, then soaking in 100% ethanol for 5 min and air-drying works pretty well. Store in an sealed container with dessicant to avoid re-hydration in humid enivornments
Jhoti,
Yes, You can store your comet slides after doing the electrophoresis, neutralizing and washing.. store in an sealed container and when you need it just rehydrate and stain
We also store the slide as described by Christopher W Theodorakis. Immerse slides in 96% EtOH and air dry before storage. we rehydrate before staining.
It is similar for us. We fix the films for minimum 1.5h in 96% EtOH and air dry them. After that, I store them in a common foto album, the ones with plastic pages and pockets for the single fotos (dry and dark). You can store them like this up to 6 months (maybe even longer).
After electrophoresis, we wash the slides 4x in water (10 min each) and then immerse them in 100% EtOH for 10 mins. After removing the EtOH, we allow the slides to air dry at RT overnight in slide boxes. Storage of the slides is good as long as they remain at RT. I have had experience to keep slides even after staining. I remove all liquid by tilting the slide and then placing them back in the slide box for storage at RT. The next day, the previous stain is still faint, but just add additional stain and you will be able to observe your results. Storage can be long term as long as the environment is dry and ambient.
After preparing my slide with a first layer of NMAgarose, I will add my sample (mixed in LMAgarose). Is it necessary to add another layer of NMAgarose ontop? I'm worried that if I do add this extra layer my stain will not penetrate my cells.... Please offer any advice or tips!
I don't use any slide coating to perform the comet assay. We use the specialized slides from Trevigen that are made for performing the assay. I would not recommend adding additional agarose on top. This may impact your staining and therefore your results. The only suggestion I can make is to try it on a small scale with both ways and see.
We use GelBond® Film for agarose gels which are ready to use. No slide preparation necessary. Very convenient.
Another layer of agarose should not be necessary. If you do need though you probably need more dye or longer staining period.
Hi Jhoti
No, you do not need to add another layer of NMA, Just take care when removing the coverslip from the LMA layer, in order to preserve it.
All of the colegues´ suggestions are valid. In addition, I suggest you to follow the basic protocol of Singh et al. (1988) for electrophoresis and to stain your slides according to the silver stainning protocol proposed by Garcia et al. (2004), whose files are attached. You can preserve stained slides for a long time for subsequent analysis. At least, you don´t need to perform the analysis soon after the stainning process as you do with ethidium bromide or other fluorescent dye.
Good job.
Hi,
You can try this protocol:
http://www.cometassayindia.org/Protocol%20for%20Comet%20Assay.PDF
Good luck!
I think all the above suggestions are valid. We use normal slides (they are much cheaper than the trevigen ones) and we use the third layer of LMA. The stain (we use ethidium bromide) can penetrate the agar layer. As the others, we fix slides in absolute ethanol (5 min) after electrophoresis and neutralization. Fixed slides can be stored for long time (months) before staining and analysis.
For those who use CometAssay HT slides, can these slides be re-used? or are they slingle-use only???
Hi.
I also don´t add another layer of NMPA. I wash my samples three times with Tris (pH 7), each time for about a minute after electrophoresis. Then I wash them in dH2O, put into a slide box and store in a fridge until staining.
You can store your comet slides after doing the electrophoresis, neutralizing and washing.
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