Comet Assay Protocol ( Can be store for 2 weeks)

PBSLysis Solution25M Nacl- 146.1gm100mM EDTA-37.210mM trisBase- 1.2gm

Add all above ingredients to 700ml of dH2O. Add 8gm NaOH & allow to mix for 20 minutes. Adjust pH 10 using NAOH or concentrated HCL, make final volume 890ml with dH2O.Finally add 1% Triton x.10% DMSO.Then refrigerate for at least prior to slide addition.

Electrophoresis Buffer.

TBE (10X)Tris base 108gmBoric Acid 55gmEDTA 9.3gm

Dissolve in 900ml of H2O. Adjust volume 1liter. For 1X used 1:9 ratio with water.

Alkaline Electrophoresis Buffer. (300mM NaOH/1mM EDTA)

Prepare from stock solution10N NaOH (200gm/500ml dH2O)200mM EDTA (14.89/200ml dH2O) pH10

For 1X (per liter)Add 30ml NaOH and 5ml EDTA mix well

Neutralisation Buffer0.4M Tris 48.5 to 800ml adjust pH to 7.5 with concentrated HCl.

Staining solutionEthidium Bromide (10X stock 20μg/ml) 10mg in 50 ml.

ProcedurePrepare lysis solution and chill at 40°C and or on ice for atleast half hour before use.Melt LMAgarose in beaker of boiling water for 5 minutes with the cap loosened place bottle in 37°C water bath for 20 minutes. Combine cells with LMA at ration 1:10 and immediately pipette 75 μl on slidePlace slide flat at 4°C in the dark for 10 minutes (gelling time can be increased to 30 minutes)Immersed the slides in prechilled lysis solution and leave on ice at 4°C for 60 minutes(45 minutes)Tap off excess buffer from slides and immersed in freshly prepared alkaline solution. (pH>13)Leave the slide in alkaline solution for 20 to 60 minutes at room temperature in dark.

TO PERFORM TBE ELECTROPHORESISRemove slide from alkaline solution, gently tap excess buffer from slide and wash by immersing in 1X tbe buffer for 5 minutes.(2 times)Transfer slide from TBE buffer to horizontal electrophoresis apparatusPlace slides flat on to a gel tray and align equidistant for the electrodes.(10 minutes running)Very gently tap off excess TBE and dip slide in 70% ethanol for 5 minutes.Air dry samples drying brings all the cells in a single plane to facilitate observation.At this stage samples may be stored at RT in dessicator.

ALKALINE ELECTROPHORESISTransfer slide from alkaline solution to a horizontal electrophoresis apparatus Place the slide on gel tray align equidistant from electrodes.Perform electrophoresis for 20-40 minutes.Gently tap off excess of buffer by dipping in H2O and then immersed slide in 70% ethanol for 5 minutes.Air dry sample and store at room temperature.