Why is there loss of agarose disks from the CometSlide™?
Posted: Tue 17 Feb 2015 13:02Why is there loss of agarose disks from the CometSlide™?
FAQ from Trevigen
CometSlides are specially treated in order to enhance agarose binding during alkaline treatment.
Special attention needs to be paid during spreading of LMAgarose on the slides.
Generally, place 50 ul LMAgarose on the center of each well (2 well slides) and then gently spread the agar using the side of a pipette tip.
You don’t want a mound of agar in the center, because the adhesion area needs to be in contact with the agar over the entire well. Be careful not to get any LMAgarose on the red border for when it dries it increases the chances of the agar coming off or folding over.
All processing of the slides needs to be done very gently. Place the slides into solutions; never pour or decant buffer onto CometSlides.
Minimize the time the slides spend in alkaline buffer. We recommend performing the unwinding step for 20 min at room temperature and then transfer the slides into an electrophoresis tank containing 4°C buffer and electrophorese for 20-40 min depending on the unit (we recommend using our new ES unit cat# 4250-050-ES).
Other possible causes/recommendations:
Cells were not washed to remove medium before combining with LMAgarose:
The pH of the medium and carry over serum proteins, etc., can reduce adherence of the agar.
Resuspended cells in 1X PBS.
LMAgarose percentage was too Low:
Do not increase ratio of cells to molten agar by more than 1 to 10.
LMAgarose was not fully set before samples were processed:
Place slides at 4°C for 15 min to assure complete gelling of the agar.
Ensure a 0.5 mm dried ring, due to agar disc retraction, is seen at the edge of the CometSlideTM area.
FAQ from Trevigen
CometSlides are specially treated in order to enhance agarose binding during alkaline treatment.
Special attention needs to be paid during spreading of LMAgarose on the slides.
Generally, place 50 ul LMAgarose on the center of each well (2 well slides) and then gently spread the agar using the side of a pipette tip.
You don’t want a mound of agar in the center, because the adhesion area needs to be in contact with the agar over the entire well. Be careful not to get any LMAgarose on the red border for when it dries it increases the chances of the agar coming off or folding over.
All processing of the slides needs to be done very gently. Place the slides into solutions; never pour or decant buffer onto CometSlides.
Minimize the time the slides spend in alkaline buffer. We recommend performing the unwinding step for 20 min at room temperature and then transfer the slides into an electrophoresis tank containing 4°C buffer and electrophorese for 20-40 min depending on the unit (we recommend using our new ES unit cat# 4250-050-ES).
Other possible causes/recommendations:
Cells were not washed to remove medium before combining with LMAgarose:
The pH of the medium and carry over serum proteins, etc., can reduce adherence of the agar.
Resuspended cells in 1X PBS.
LMAgarose percentage was too Low:
Do not increase ratio of cells to molten agar by more than 1 to 10.
LMAgarose was not fully set before samples were processed:
Place slides at 4°C for 15 min to assure complete gelling of the agar.
Ensure a 0.5 mm dried ring, due to agar disc retraction, is seen at the edge of the CometSlideTM area.