It seems likely that something is wrong with the unwind/electrophoresis steps. Typically, slides stay in alkaline buffer for 30min before the application of current (electrophoresis).  

I agree with Doulias.something wrong at electrophoresis process, most likely electrophoresis solution is unsuficient, e.g. old solution.

Thank you very much for your attention

We let slides sit in the alkaline buffer for 30 minutes at ~4ºC before electrophoresis and used fresh solution for it.

We'll be so grateful to let us to know more possible errors that might be happened.

Sincerely

Nahid

Do you mind to provide more detailed procedure? It is unlikely the 50uM Hydrogen peroxide can induce DNA damage in whole blood since catalase inside will degrade it.

Hydrogen peroxide at 50uM does induce DNA damage in isolated cells, this is very well established

Sure, but not whole blood. Red blood cells contain large amount of catalase. Hydrogen peroxide at even 1 mM may not able to induce DNA damage of WBC in whole blood.

As others said it seems that something is wrong with the electrophoresis step. Could you give us more info about the buffer solution and the current (V, mA) you are using? I used to set the current at more or less 1 V/cm taking into consideration the total lenght between electrodes in the chamber, and at 300 mA.

Thank you so much for your attentions…

-The procedure was almost done according to the attached protocol.

-Electrophoresis Buffer was Prepared freshly, ensured pH to be >13, refrigerated prior to use and consisted of:

NaOH stock solution (10N) … 30ml (Prepare every 2 weeks)

EDTA stock solution (200mM) … 5ml (Prepare every 2 weeks)

dH2O…  q.s. to 1000m

-The voltage and the current were adjusted to ~ 22 V and 300 mA respectively, by raising or lowering the buffer level and the length between electrodes was ~ 20 cm.

We’ll be glad to know your suggestions about our experiment and their defects.

Also we’ll be so pleased to know your recommendation about the concentration of H2O2 or other agents which can be used as positive controls.

Sincerely

Nahid Ahmadi

For lymphocyte: 50uM hydrogen peroxide is good enough as positive control.

For whole blood: you may consider to use UV light instead.

Thank you so much for your consult.

Would you please let me know more details about UV-induced genotoxicity procedure(Device, exposure duration,...)?

The slides are subjected to oxidative stress by irradiating the slides with UVB at 6cm distance from the source for 2 min, after removing of the cover slips. The Spectroline UV light box can be used.

Hi Nahid

If you want a great positive control for your alkaline comet assay, why don't you try etoposide?  25 microM etoposide (probably less for lymphocytes) will cause SSBR and DSBR within 1 h and plenty of tails to look at!  My (limited) experience with H202 is that it is rather finicky (and a weak and non-specific DNA damaging agents)...and depending on your exposure time, recovery time, cell type etc..you may not see much. To what end are you using the comet assay exactly? Death or DNA damage?

Not familiar with lymphocytes (likely quite sensitive) but don't  assume H202 to give you much measurable results...comet assays sensitivity is limited and would consider other options to look at DNA damage specifically (especially with H202).  My feeling is that H202 may be killing your cells via lots of ROS sensitive pathways (not DNA damage necessarily) and your tails will reflect dying cells not DNA damage.

Good luck!

Sorry for frustrating comments given by some others so far. If anyone could read and check the pics carefully, it may be clearly understood that the problem is at the electrophoresis (EP) step, because there seem no MOVING shaped comets, H2O2 or distilled water is not the matter in all treatments. I advise again to Nahid. Follow the protocols and apply carefully, first make sure expiry date for chemicals. Prepare EP solution just one day before the practical, keep at +4C at the fridge. During the EP, do not let the EP machine and solution get heated, for example apply ice bags under the machine if heat occurs.  Comet always works, even you will see moving comet shapes in distilled water treatments. Four your experience, apply the comet work only using distilled water for your practice, for example get blood from yourself or from a mouse or from a hen at the laboratory instantly, just apply the protocol without any control group. As a result I am sure you will be happy. This is just my opinion. Best wishes.

To clarify: healthy cells have negligible comet moments...so yes, if you don't damage them somehow, DNA will not migrate out of the nucleus, and you will get no tail ( by the way your mag is on the low side...you should get high magnification to quantify properly...and WAY more cells per field of view).  I seriously doubt the problem is with running conditions.  All that stuff about temperature etc....has its place for reproducibility (moment values etc..), but we never had issues (except for the agarose detaching off slides when temperature is on the high side) using conventional electrophoresis tanks.  

Good luck

Seeing the photos I consider that your problem is rooted in the operation of electrophoresis. Apply the protocol as each step is specified, taking care in temperature and reagent preparation. Best regards

Dear professors,

Thank you for responding to my inquiry. Your valuable and constructive inputs are greatly appreciated.

sincerely,

Nahid Ahmadi

The manual techniques as given by Tice And Dhawan in separate papers works well. The slide is coated with NMPA and drdried.then blood is mixed h low mp agar at 37  deg and spread on the slides. LYsed in chilled buffer and then put for electrophoresis i

Dear all,

Recently comet assay has been set up in our labs.

Thank you so much for all help and advice.

Sincerely,

Nahid Ahmadi