when I run the comet assay slides in lysis buffer pH 10 for 30-60 minutes, I found some of the slides lose the agarose and some completely dissolved form the slide.
first I was used commercial OxiSelect™ 96-Well Comet Assay Kit from Cell Biolabs.
I followed them instructions for running the test, but, unfortunately, most of the wells has lost the agarose.
then I repeat the test after preparing the slides using different procedures as follow:
1. 0.5% agarose ( 1:10 combined cells with agarose), 50 micro.l. was putted on the slide and then the first layer of 200 micro.l. was done, left until agarose solidified and then added 50 micro.l. of combined cell with agarose and left until solidified then added 200 micro.l. of agarose as second layer until solidified finally I was put the slides in lysing solution for 30-60 minutes.
2. 0.75% agarose ( 30 micro.l. cell suspension + 270 micro.l. of liquified agarose 42 C). 120 micro.l. of combined cells with agarose loaded on the slides and covered with coverslip until solidified and removed the coverslip and added 200 micro.l. of agarose as the second layer, covered with coverslip until solidified and then removed and put in lysis solution.
Note: I am using tissue samples of rats' ovary.
this is my problem, please I need your help.
Dear Ali,
I'm not familiar with OxiSelect™ 96-Well Comet Assay Kit, so maybe my answer will not be relevant to you...
According to my experiences with comet assay, problem is probably not the dissolution of the agarose gel, but poor adhesion of the gel to the microscope slide. If adhesion of the gel is weak, this can result in slipping the gel of the slides. Here are some suggestions:
- Preparation of comet assay slides is usually made on ice and this can results in water condensation (forming of water droplets on microscope slides). If you spread agarose gel onto wet slides, the adhesion will be weak.
- You should put prepared slides into the lysis buffer slowly and carefully.
- To increase adhesion, you can use fully frosted microscope slides instead of clear ones (nevertheless, I prefer clear slides, pre-covered with 0,5% agarose, one day before use).
Hope this helps.
Veno is spot on, I fully concur with his comment. In addition you have to ensure you use the correct slides. Ensure you are NOT using polished slides as I found that the agarose gels only weakly attached to those slides.
Thank you very much Veno and Naim for your advices. Actually, I am using fully frosted microscope slides, and I prepared the slides on ice, but the problem I use agarose at 42 C degree when i put on the slides after i warm the slides to 37 C degree, then i put the slides on the ice until solidified and then continue the procedure.
Try pre coating the slide with NMA the previous day and following application of LMPA allow it on ice for a little longer duration. Some times I have noticed that, even with increased pressure to remove the cover slip, the agarose seems to slip away during electrophoresis.
thank you very much dear professors, your opinions are highly apreciated
I used to flame the slides by dipping them in alcohol before first layer of NMA this removes the particles and grease if any that has adhered to slides during manufacturing. Thus helping better adherence of agarose
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