How do you adjust the base layer thickness of agarose gel when electrophoresis in a comet assay?
When I was doing peparation of Slides, dip conventional slides and wipe underside of slide and just lay the slide on a flat surface to dry.
I think it will be a little bumpy even if it looks flat and thin to the eyes.
In this situation, does the thickness of base layer affect electric motion?
Hello Jae-I Moon
The quality of slides that you use in comet assay is important. I suggest you use frosted slides because they have a rough surface that will help the agarose layer to attach in a better way forming a uniform layer when you prepare the slides.
Make sure that your slides are not greasy or dirty or else the agarose will not set on the slide and will be easily removed during the process of lysis or electrophoresis. So before you carry out the assay you should wash the slides with soap water using a brush followed by a dip in methanol and then flame the slides to remove the methanol. Then you can layer the agarose on the slide. After layering the gel you can flatten the gel evenly by placing a cover slip of a similar size to that of the slide over the gel and incubate at 4˚C for 10 mins for the agarose to solidify. After solidification you can remove the cover slip. You will get a thin uniform layer of agarose on the slide. Having a uniform gel coat on the slide will help in better anchorage for the subsequent agarose layers that will be layered over it.
The electric motion is least affected as rightly mentioned by Eleni.
Thank you.
The thickness of the base layer of the gel would obviously affect the mobility.. I use the simple blood smear technique that we do to prepare a DLC blood smear slide.
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