I'm standardizing alkaline single cell gel electrophoresis (comet assay) for ionizing radiation induced DNA damage. Out of the 12 slides, I barely get 3 or 4 slides with the agar coating while the others' coating slips after electrophoresis. I'm incubating in lysis buffer(pH =10.0) for 1 hour then 20 min unwinding in electrophoresis buffer pH=13.6 followed by 20 min run at 25 V/300A 1V/cm. As soon as I neutralize the slides (pH=7.6) the agar coating slips. I've precoated the slides with 1%agar and then added 0.5%low melting agar with cells.
The slides which did not slip gave good results, stained with Propidium Iodide. How can I increase the output and prevent slipping?
Hey!
One thing that I do and works for me, is to incubate at 4C for 30min my slides once I dropped the cells mixed with the low melting agarose. This helps the agar to dry a little bit, you will see a ring forming around your agarose drop. Another thing is that instead of pouring the different buffers and solutions on top of the slides, I like to add these to the container first and then place my slide in the solutions.
Hope it helps.
Thanks Paula,
I do keep the slides at 4°C after adding the LMPA+cells. I'll add the buffers first and then the slides.. Thanks I'll update asap. Could it be a problem in cleaning the slides? I use alcohol and tissue to clean the slides. Perhaps I need to flame the slides as well?
y not sip out the reagents first from the drain tray with a 10 ml pipette or likewise and then take your slides out carefully and horizontally with the help of forceps or something .
Hi!
I would like to know how have you stored your precoated slides?
Because I was having the same problem during this summer and we found one solution for the problem. Usually we kept the precoated slides at the room temperature covered from light but when we changed our protocol, having precoated slides stored at the freezer (about - 20 °C) the problem disappeared. Take the slides from the freezer about 0,5-2 hours before starting the comet assay.
I can´t say why this worked but maybe it has something to do with the humidity. But I recommend you to test this trick if you haven´t done this way before.
Hope it helps!
Hi,
after pre-coating I dry the slides in a 45 C oven so that they dry up really well.
More importantly, after rising the slides horizontally from the electrolysis tank I have neutralized the slides just by putting them horizontally on a tray and adding 800 µl neutralizing buffer on top of each plate for 5 min. After 5 min I tip the slides and let the buffer absorb into the laboratory tissue I have put under them. This is repeated three times. I have also used neutralizing in a Coplin jar, but it sometimes has led into detouching, this virtually eliminates it.
The freezer tip seems worth testing, but if nothing else works, this will.
Hope some of the answers help you!
Hi
You should be Get a smooth surface at the stage of preparation of coating layer from agar and dry well slides, pour the Buffers slowly from one side and inside the tank containing the slides so that it is not poured on the slides directly , then incubation time discard slowly buffers on slides.
Thank you all,
@Mikko I usually precoat the slides and keep them for 24 hours at 4 deg C horizontally. I'll definitely try the -20 deg C tip. It might work. Thanks a lot.
@ Anna - I always treat the slides horizontally as you have described. If the freezer tip doesnt work, I'll dry the slides at 45 deg C. I have dried the agar coated slides but not for comet assay. Do i store the slides at room temp. or 4 deg C after drying? Also, after drying, the agar dries and appears to have crystallized, will that affect the experiment? Do i need to rehydrate the agar?
@ Beheshte - I do try and get a smooth coating at the pre-coating stage. The slides which appear uneven, I either discard them or re-coat them.
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