Protocol Online logo
Top : New Forum Archives (2009-): : Tissue and Cell Culture

agarose sterilization - (Oct/14/2013 )

Hi all, I have been attempting some soft agar assays but have had contamination the second day after plating.  After 3 attempts (and nothing else getting contaminated- other assays, cell cultures, etc) I am convinced it is not my technique but that the agarose is contaminated and needs to be sterilized.  I am using low melt temp agarose (only opening it under hood) and dissolving it in sterile DMEM in a 65 deg water bath.


So my question is, how can I sterilize the agarose?  Can I filter it, and if so what pore size (or will the agarose just clog up the filter?)  Or can I autoclave it (or is this too hot and will it break down the agarose?).  Any help would be greatly appreciated, thanks!


On way to get around this is to make up double % agarose in water (e.g. if you use 1% for your overlay, make up 2%) autoclave.  Also prepare 2x medium.  When you want to do your overlays, mix the 2x agarose and 2x medium 1:1 to give correct concentrations of each.


you can try microwaving it. The time will depend on the volume, but you can google it to see how others do it.


hi .yes you can autoclave or boil agar autoclave in121c for 20 min based on culture of animal cell page148 sterilization of liquid i taked apicture of this page for help to you this page include 30 other materials for strilization 

Attached File