In several repetitions that I performed, there is no tail formation on the nucleoids, even using different mutagens / damage inducers (MMS - 200 uM; H2O2 - 400 uM and Cisplatin - IC50 value). I have already performed the comet assay with hepatocytes from C57BL6 mice and with A-375 and B16-F10 cell lines and this problem still persists.
The running buffer used is: -H2O (Deionized water) -NaOH 10N -EDTA 200mM pH> 13
25V / 300mA - 20 minutes
**During electrophoresis, there is the formation of bubbles in the electrophoresis buffer (I believe the electrophoresis chamber is working).
The concentration of agarose used was:
Normal agarose - 1% or 1,5% (v/v)
Low Melting Point Agarose - 0,5 % or 1% (v/v)
What could be happening?
I appreciate all the help available
*Attached are some images of the nucleoids that I obtained in my last experiment (A-375 Cell line).
What are your experimental parameters? How long do you keep cells in damaging agents, is there time for cells to repair the damage? I did some experiments recently with cisplatin and almost all DNA was fragmented so that is what I would expect to see on comet as well.
Hi Arthur. I hope everything is fine with you.
Your experiment seems to me all right. When there is no tail formation, we normally infer that there is no DNA damage. As for the formation of "bubbles", my group called them "Ghost", and these should not be included in the analysis, they are discarded.
I believe that there may be problems with the preparation prior to cell lysis. The cells must be prepared carefully when pipetting, do not forcefully dissolve, so that the membranes do not break before the process of lysis and electrophoresis.
I hope I've helped.
Dear Arthur,
Also give a try to human lymphocytes untreated and treated with H2O2. First you should try to see the normal cells with normal shape. Are these hepatocytes?. Use 0.8 % NMA and 0.7 % LMPA. Don't use hot LMPA. Also check another electrophoresis unit and power supply, if available. Otherwise movement of bubbles show the working / current flow of electrophoresis unit. try 30 V, 300 MA and 30 min. make fresh solutions and check for pH to be alkaline. Keep the slides in lysis solution for over night. Also use chilled solutions (lysing and electrophoreis). Leave the slides for 15-20 min in electrophoresis buffer before the start of electrophoresis step. It might work In Sha Allah!
Best of luck! :)
LMPA and RMPA concentrations is looking high. Reduce its concentrations....
As my Ph.D student Ajmal khan clearly explain every thing. Confirm first the treated and untreated hepatocytes under light microscope. Use milli q water for solution preparation instead of deionized water.PBS solution must be ca/ mg free.
Lysis buffer may not be working. What did you use as lysis buffer and how long did you treat the cells with it?
Hi Arthur
Maybe the pH is too high. Because at above pH 13, only basic marked positions will be observed. Try it again at 12
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