When you add agarose, add cover slip on the top of laid agarose. This will make the liquid gel flatten b4 it dries. Later you should gently remove the coverslip to further use the slides in experiment

I used 0.75% LMA, and 1:1 dilution (LMA : cell suspension). It gives better result. You take 200 µl of the above suspension for second layer slide preparation.  immediately place the coverslip and maintain the slides at 4 ºC for 10 min. Then remove cover slip and place the slides in lysis buffer. The lysis treatment is depend on what cell you used and lysis temperature, we are giving overnight at 4 ºC.  For electrophoresis 0.74 V/cm and 330 mA current is sufficient.

All the best

Put a scotch tape on two ends of Slide then add your desired percentage of agarose on to it then place one more slide without scotch tape on the  slide . Then snap freeze the same and after half an hour remove the top slide.This technique is also used to prepare live yeast cell imaging

You can watch this youtube video for more technical assistance

https://www.youtube.com/watch?v=ZrZVbFg9NE8&hd=1

Hi.

I also had the same difficulties when I started running the assay. 

The gels came off as soon as i put them in the lysis buffer or sometimes at the electrophoresis stage.

However, I found a way out with the help of a senior colleague.

I used the 3 window diagnostic slides (it doesn't matter even if it is the conventional slides you use) and coated each window with 15ul of 0.75% LMP Agarose first. I dried these in the incubator at 37 degrees for 30 minutes. Thereafter,  i coated again with 15ul of 0.75% LMPA and allowed to dry at least for 24hours before use. I think the longer you keep them the better. Perhaps prepare your slides between 2-5 days before your assay and leave them in the incubator. 

From experience, i saw that when i did this, it didn't matter if i kept the slides coated with cell suspension in lysis buffer for 1 or 24 hours, the cells stuck throughout the process and the results were fine. Depending on the number of samples though, you can incubate your cells coated slide in the lysis buffer overnight (not more than 24hours) on ice (i also incubate them at  4 degrees in addition to putting the slide racks on ice).

Hope you find this useful.

All the best.

We routinely use the following protocol for comet assay in our lab. You may have to adjust it for your particular experiment i.e. if you do a different treatment other than radiation but we don't have any problems with the slide preparation.

Comet assay solutions

Agarose

Normal melting point agarose (1%) in dH2O

Low melting point agarose (1%) in PBS

Lysis Buffer

Solution of dH2O (800 ml) containing NaCl (146.1 g, 2.5 M), EDTA disodium salt (37.2 g, 100 mM) and Tris base (1.2 g, 10 mM). set to pH 10.5 by the addition f solid NaOH (8 g) and 10 M NaOH dropwise. Adjust to 1 L and store at 4C.

Prepare complete spllution just rpior to use by additing a mixture os 1 ml DMSO and 1 m triton-X-100 to 98 ml cold lysis buffer.

Electrophoresis buffer

300 mM NaOH, 1 mM EDTA amd 1 % DMSO, prepared just prior to use

NaOH (100 g in 500 ml, 5M) and EDTA (14.8896 g in 200 ml, 200 mM)

Add 60 ml NaOH, 5 ml EDTA and 10 ml DMSO to 925 ml dH2O. Check pH>13 and store at 4C until required.

Neutralisation buffer

Tris-base (12.11 g in 100 ml, 500 mM), pH 8.0

Staining solution

Dilute SYBR Gold 1 in 10,000 in dH2O, pH8.0

Comet assay protocol

Prepare slides by adding 800 µl molten normal melting point agarose to a slide, add a coverslip and leave agarose to set. Remove coverslip and air dry slides overnight.

Trypsinise exponentially growing cells and dilute to 1x105 cells/ml. Add 250 µl cell suspension per well of a 24-well plate on ice to prevent repair and adhesion. Irradiate cells.

Add low melting point agarose (37°C) to cells prior to adding 1 ml to the NMPA cpated slide. Add coverslip and place on ice tray for 5 min.

For repair studies, place slides in a humidified chamber at 37°C.

Remove coverslips and place in coplin jars containing fresh cold lysis buffer and lyse cells for at least 1 h at 4°C.

Transfer slides to electrophoresis tank and cover with electrophoresis buffer (4°C) and allow DNA to unwind for 30 min.

Electrophorese at 25 V for 25 min, adjusting the current to 300 mA by adding or removing buffer.

Remove slides from tank and cover with 1 ml of neutralisation buffer for 5 min. Repeat twice.

Rinse the slides in dH2O and allow to air dry over night.

Rehydrate slides in dH2O for 30 min and add 1 ml of SYBR Gold to each slide for 30 min. Remove excess stain and rinse in dH2O.

Allow slides to air dry in the dark for storage and analysis.

All the best.

You might want to check Nishi et al., NCB, 2014 (DOI: 10.1038/ncb3028)

It describes in detail the experimental procedure and it works very well.

Good luck!

Thank you very much Pamela Reynolds for the protocol

Hi

You might want to check "PROTOCOL FOR THE SINGLE CELL GEL

ELECTROPHORESIS / COMET ASSAY FOR RAPID

GENOTOXICITY ASSESSMENT " prepared by Alok Dhawan, et al. 

Hope you find it useful.