Cells in LMAgarose not remain attached to the CometSlide?
Posted: Tue 17 Feb 2015 15:13
What would cause the cells in my LMAgarose to not remain attached to the CometSlide™?
FAQ from Trevigen
- Electophoresis solution may have been too hot - Control temperature by recirculating the electophoresis solution or performing the assay at less than 20ºC.
- Cells were not washed to remove medium before combining with the LM agarose. - The pH of medium and carry over serum proteins, etc., can reduce the adherence of the agarose. Resuspend cells in 1X PBS.
- Agarose percentage may be too low - Do not increase ratio of cells to molten agarose by more than 1 to 10.
- LMAgarose was not fully set before samples were processed. - Ensure 0.5 mm dried ring due to agarose disc retraction is seen at the edge of the CometSlide™ area.
- LMAgarose unevenly solidified on the slide. - Spread agarose with the side of a pipette tip to ensure uniformity of agarose disc and better adherence.
- LMAgarose was not completely melted. - Completely solubilize the agarose berofe transferring to a 40ºC heat block.
Rinse steps were too harsh. LMAgarose after alkali treatment may detach when rinse solutions (e.g. dH20, EtOH) are poured over slides. - Gently submerge slides into pipette box lids or other suitable tray to perform rinses.
FAQ from Trevigen
- Electophoresis solution may have been too hot - Control temperature by recirculating the electophoresis solution or performing the assay at less than 20ºC.
- Cells were not washed to remove medium before combining with the LM agarose. - The pH of medium and carry over serum proteins, etc., can reduce the adherence of the agarose. Resuspend cells in 1X PBS.
- Agarose percentage may be too low - Do not increase ratio of cells to molten agarose by more than 1 to 10.
- LMAgarose was not fully set before samples were processed. - Ensure 0.5 mm dried ring due to agarose disc retraction is seen at the edge of the CometSlide™ area.
- LMAgarose unevenly solidified on the slide. - Spread agarose with the side of a pipette tip to ensure uniformity of agarose disc and better adherence.
- LMAgarose was not completely melted. - Completely solubilize the agarose berofe transferring to a 40ºC heat block.
Rinse steps were too harsh. LMAgarose after alkali treatment may detach when rinse solutions (e.g. dH20, EtOH) are poured over slides. - Gently submerge slides into pipette box lids or other suitable tray to perform rinses.