Dear Colleagues, I'm struggling with the comet assay - I can't see the expected comet shape.
I work with three isogenic lines, including the control line, monoallelic, and biallelic mutant. I do not perform any treatment (except for the positive control with etoposide).
My issue is the fact I don't know whether my cells are lysed or I have a problem with electrophoresis. As you can see in the photos of my first attempt (bottom) there are no comets except for the positive control. In the second attempt (top) with prolonged lysis, the results are similar, but I can see some comets in the Mut_2, however, their shape differs within the same slide.
My clue is that it might be due to electrophoresis because I do it in the standard BioRad tank where the classic DNA electrophoresis is performed (attached). Please find below my detailed protocol. I'd be grateful for your help.
1. Before harvesting the positive control positive control was stimulated with 10uM etoposide for 1,5h
Low melting agarose was melted in the incubator and aliquoted 300 uL for each sample (4 total) and stored at a thermal block at 37*CMicroscope slides preparation: The slides were coated yesterday in 1g agarose + 100 mL dH20 by drawing slides (~10 sec in this solution. The bottoms of the slides were cleaned and slides were stored at RT for further use on the same day.The electrophoresis buffer was prepared according to the instructions below (0,5 L): Prepare separate stock solutions: 5 M NaOH (100 g NaOH in 500 ml dH2O), 200 mM EDTA disodium salt (C10H14N2Na2O8·2H2O; 14.89 g in 200 ml; no pH adjustment necessary). To prepare electrophoresis buffer (final composition: 300 mM NaOH, 1 mM EDTA, and 1% DMSO), add 60 ml 5 M NaOH, 5 ml 200 mM EDTA, and 10 ml DMSO to 925 ml cold dH2O; check that the pH is >13 (using a pH strip) and store at 4°C until required.The lysis buffer was: Prepare the ‘premix’ by dissolving 146.1 g NaCl (final: 2.5 M), 37.2 g EDTA disodium salt dihydrate (C10H14N2Na2O8·2H2O) (final: 100 mM), 1.2 g Tris base (final: 10 mM), and 8 g solid NaOH in 800 ml dH2O. Once all the solids have dissolved, adjust the pH to 10.5 by the dropwise addition of 5 M NaOH. Adjust the volume to 1 L by adding dH2O and store at 4°C. To prepare a ‘complete solution’ of lysis buffer immediately before use, add 1 ml DMSO and 1 ml Triton X-100 to 98 ml cold lysis buffer.Cells were harvested, spun, and suspended in 25 uL PBS and mixed with agarose.Immediately pipette 50 µl onto two spots of prepared slides located on a tray. Two technical replicates for each sample were placed in one microscope slide (2x 50 uL). Each droplet containing agarose+cells 25x25 mm coverslips was placed, distributing agarose with cells (you can press the coverslip gently).Slades were placed at 4°C in the dark for 10 minutes and then coverslips were removed.Slades were immersed at 4*C in the working Lysis Solution for 60' or overnight.Then the slides were immersed in 4°C 1X Alkaine Electrophoresis Buffer for 30 minutes.After that, the electrophoresis was performed at 21V 30'.Slides were immersed twice in dH2O for 5 minutes each, then in 70% ethanol for 5 minutes.Samples were dried at 37°C for 10-15 minutes.100 µl of diluted SYBR® Green (10 000x) (Cat 50513 Lonza) (Diluted in PBS) was loaded onto each sample and incubated at 4*C for 30 minutes. Even dye distribution was ensured by placing the piece of parafilm covering the whole slide.The excess SYBR was removed by briefly rinsing in waterSlides were dried completely at 37°C
