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agarose sterilization

soft agar contamination

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3 replies to this topic

#1 Christi

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Posted 14 October 2013 - 08:03 AM

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!



#2 bob1

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Posted 14 October 2013 - 01:29 PM

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.



#3 CellApplicationsInc

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Posted 25 October 2013 - 01:34 PM

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



#4 antitoxin

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Posted 28 October 2013 - 02:06 PM

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 

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