Dear Salma,

We use regular microscope slides frosted one end (size 25 mm × 75 mm).

A. 1% NMA in distilled water is prepared for precoating slides (heat the solution on bath or carefully microwave bottle at low power for short intervals to avoid vigorous boiling of the agarose and ensure that all agarose is dissolved – it becomes transparent) (min 0.45 in the movie)

- label the slides on frosted end using a pencil, not a pen;

- dip each slide into molten agarose up to half frosted end and wiping one side clean (min 2.00 in the movie) (we keep the vial with agarose at ~ 90°C in water bath);

- allow agarose thin film to air-dry on an even surface. Slides can be prepared ahead of time and stored with desiccant (assure a good adhesion of agarose).

 B. Prepare 1% LMA in phosphate-buffered saline (without bivalent cations) – after melting, LMA is cooled and kept on water bath fixed at 37°C

 C. Prepare cell suspension with a density to about 2 × 104 cells/ml in phosphate-buffered saline.

 D. Pipet 0.4 ml of cells into Eppendorf tube (2 ml capacity) (keep on ice until mix with LMA).

 E. Add 0.8 ml 1% low-gelling-temperature agarose at 40°C.

 F. Mix and rapidly pipet 1.0 ml of cell suspension onto the agarose-covered surface of a pre-coated slide; avoid producing bubbles.

 F. Allow agarose to gelling for about 2 min before submerging in lysis solution.

For more information visit: https://www.youtube.com/watch?v=Nu-FEad49P8

http://www.nature.com/nprot/journal/v1/n1/pdf/nprot.2006.5.pdf.

Good luck!

Gabriela

https://www.youtube.com/watch?v=Nu-FEad49P8

http://www.nature.com/nprot/journal/v1/n1/pdf/nprot.2006.5.pdf.

Dear Salma,

We use regular microscope slides frosted one end (size 25 mm × 75 mm).

A. 1% NMA in distilled water is prepared for precoating slides (heat the solution on bath or carefully microwave bottle at low power for short intervals to avoid vigorous boiling of the agarose and ensure that all agarose is dissolved – it becomes transparent) (min 0.45 in the movie)

- label the slides on frosted end using a pencil, not a pen;

- dip each slide into molten agarose up to half frosted end and wiping one side clean (min 2.00 in the movie) (we keep the vial with agarose at ~ 90°C in water bath);

- allow agarose thin film to air-dry on an even surface. Slides can be prepared ahead of time and stored with desiccant (assure a good adhesion of agarose).

 B. Prepare 1% LMA in phosphate-buffered saline (without bivalent cations) – after melting, LMA is cooled and kept on water bath fixed at 37°C

 C. Prepare cell suspension with a density to about 2 × 104 cells/ml in phosphate-buffered saline.

 D. Pipet 0.4 ml of cells into Eppendorf tube (2 ml capacity) (keep on ice until mix with LMA).

 E. Add 0.8 ml 1% low-gelling-temperature agarose at 40°C.

 F. Mix and rapidly pipet 1.0 ml of cell suspension onto the agarose-covered surface of a pre-coated slide; avoid producing bubbles.

 F. Allow agarose to gelling for about 2 min before submerging in lysis solution.

For more information visit: https://www.youtube.com/watch?v=Nu-FEad49P8

http://www.nature.com/nprot/journal/v1/n1/pdf/nprot.2006.5.pdf.

Good luck!

Gabriela

https://www.youtube.com/watch?v=Nu-FEad49P8

http://www.nature.com/nprot/journal/v1/n1/pdf/nprot.2006.5.pdf.

Though I completely agreed with Gabriela

i would like to share my experience

As said we need frosted slides for comet assay

I pour 100 micro liter of 1 % NMA dissolved in PBS on the frosted slides and covered with cover slip for some times in cold. It gives you thin bed. (Remove the slide after solidification)

Then i mix the cell suspension with 0.8% LMA (@ 40 C) and then rapidly apply onto the first bed (NMA) and allow it for gelling

Then 0.8% LMA is prepared as third layer.

Good luck

T he question you have asked is.

1. you can flatten the agarose by using another slide.

2. I am using 120 ul for two layers and 100 ul for the last one.

3. Putting them on ice to solidify asap

4. I prefer two layers then the last one

Dear Salma,

This is the protocol I use

Preparation of slides

1st Cover

The doors have to be glazed to maintain the solidity of the whole and allow the layers to remain firm.

The slides are covered with 110 microliters at 1% normal melting point agarose (NMA) dissolved in buffer (1 x PBS) at 45 ° C. It is then covered with a coverslip and held at 4 ° C for 10 min to solidify the Agarose.

2nd Cover

Place a culture volume according to viability, place in an eppendorf and centrifuge at 15000 rpm for 5 min.

            In Vitro Studies: approximate volume of 100 microliters (80-200 according to

            toxicity)

            Studies with populations: 60 microliters

            Prepare two replicates for each concentration or individual

Remove supernatant

After 10 minutes of standing the doors cold, carefully remove coverslips

The blood pellet (between 1000-500000 cells) is mixed with 85 microliters of LMA (low melting point agarose, important not to boil) at 0.7% and at 37 ° C. The cell suspension is rapidly pipetted onto the first layer of Agarose.

Place a coverslip and keep the doors at 4 ° C for 8 min.

3º Cover

Remove the coverslip carefully after 8 minutes

Put 85 microliters of 0.7% AML at 37 ° C above the second layer

Quickly place another coverslip and put again at 4 ° C for 8 min

Lysis phase of cell membranes

Remove the coverslip carefully

The doors are immersed, placed in a grid, inside the tray containing the cold solution of lysis and prepared in the moment (fresh-prepared). This solution will consist of:

89% of the cold membrane lysis buffer (prepared above and left cold). 1% Triton x 100

10% DMSO

Keep the doors immersed in this solution in cold (4 ° C) and in the dark for at least one hour (the time can be variable without exceeding one night).

Electrophoresis phase

Best regards

I am not getting a good 1st layer of NMA. After I dip the slides up to half the frosted end, gently remove the slide from the coplin jar, wipe the underside of the slide and leave to air dry. Within an hour, I find the agar layer starting to shrink and no longer coating the slide as it was at the start of the air drying step. What could I be doing wrong please?

Hi researchers,

I have 2 major problems about comet assay.

first problem, after 1 day dry period  I get the slide. is there any suggestion about the problem ?

how can I solve the problem ?

second problem

adherence problem about the NMA  after lysis or electrophoresis

Comet assay

The comet assay has been adapted for that originally proposed by Singh et al., (1990). Part frosted microscope slides were pre coated with a thin layer of 1% low melting point agarose (LMPA) and allowed to dry for 1hr at 37°C. The slides were then placed on a bed of ice to pre-cool them before adding an agarose sandwich. 170µl of 1%LMPA was placed onto the slide and immediately covered with a coverslip. Whilst this was setting, 170µl of the suspended cells were mixed 1:1 with 2% LMPA. The cover slip was then removed from the initial agarose layer. 170µl of the agarose/cell mix was placed on top of the first layer and again covered immediately with a coverslip. Once set, the coverslip was removed and a final layer of 170µl 1% LMPA was applied to the existing gel to form an agarose sandwich, with the cells in the middle layer. The final coverslip was removed, once the gel had set. For each cell sample, 2 slides were prepared. At this point the lights were switched off and the rest of the assay was performed using indirect light.

A pyrex tray containing 500ml of ice cold lysis buffer was placed onto a bed of ice and the slides gently lowered into this. After 1hr the slides were removed and washed  by placing them in 500ml ice cold PBS and allowing them to sit for 15min. A horizontal electrophoresis tank was surrounded by ice and filled with 2L of ice-cold alkali buffer. The slides were then gently lowered into the tank and levelled with the frosted end to the right. The tank was aligned using a small spirit level to ensure electrophoresis occurred straight through the slide. The slides were left for 30min to allow the DNA to unwind in the alkali buffer. Electrophoresis was then performed at 22V (500mA) for 30min. Next, the slides were removed and rinsed in 500ml of ice cold neutralising buffer for 10min and then in 500ml of ice cold phosphate buffered solution (PBS) for 15min. If the slides were being analysed on the confocal microscope within 24hr they were stored in a moist, dark environment at 4°C.

What are the electrophoretic factor which affects comet assay.