Comet Assay

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Aquaglow
Aquaglow's picture
Comet Assay

 Hi!
I am performing comet assay for the first time. My problem is that I have treated normal isolated lymphocytes with 100 uM H2O2 but the cells are not showing any comets. they are not showing any halos either. To me it seems like the cells are not lysed although I have given them lysis time of 24 hours. I haven't added SDS in the lysis buffer only Triton-X 100. Also I haven't used PBS in the agarose solution. Please can anyone tell me where I am doing wrong? ALso please can anyone tell me what is the orientation of slides in the gel tank. Is it horizontal or perpendicular to the electrodes. I am new to CA so any help is greatly appreciated. 

Damilola
Damilola's picture
 I have a similar problem

 I have a similar problem with a lack of comets on my h202 treated lymphocytes. I wondered if you could please confirm my calculations for me please. I am thinking maybe this is my problem

1. I initially made a 9.79mM solution form 30% w/w h202 

   by adding 1ul to 999PBS.

2. Then i added 2.04ul of this to 200ul of isolated lymphocyte solution to get 100uM h202.

Does this sound about right please?

If I find anything that mgith be the problem,
I will post it here for you.

D

g a
g a's picture
Dear Damilola

Dear Damilola

Check your calculations.

30% solution of H2O2 implies 30% H2O2 and 70% water. Density of H2O2 is 1.44g/Cucm
We Can calculate the mass of H2O2 that is present in a litre of this solution bu using formula

Density= Mass/ Volume
therefore, .30 x 1.44 = .432 kg or 432g
MOl wt of H2O2 is 34
 So number of moles present in 432 grams will be 

432/ 34= 12.705 or approx 12.71 moles

So Molarity of 30% H2O2 is 12.71 moles

For getting desired concentrations you need to use Molarity equation

M1V1 = M2 V2

Using Yr calculations, 

1x12.71 =M2x 1000 
or M2 = 0.01271M or 12.71mM

The value that you have mentioned here is 9,79 mM which is incorrect. So probably thats where ur flaw is.

Rectify it.

g a
g a's picture
 Dear Aquaglow

 Dear Aquaglow

It is recommended to use PBS which is Ca and Mg free for comet assay.
Here is a link for the Protocol and troubleshoot from a commercial supplier.

http://www.amsbio.com/protocols/Comet_Assay.pdf