questions about soft agar assay - (Apr/14/2010 )

I have been trying to do soft agar assay, which is a pain to me, especially when I have several cell lines. I have a couple of questions. I hope experts here can help me out.

Question 1: I saw people use agarose on some protocols. Is that the same thing as agar?

Question 2: how many times I may reheat the agar without affecting the experimental result? When I have several cell lines, the remaining top agar always set after I was done with the first cell line. I am wondering if I may reheat it again and again.

Question 3: after adding the top agar with cells onto the bottom agar, should I put the plates at 4C to solidify or just 37C incubator?

Question 4: what do you guys use to visualize the colonies? I have seen crystal violet or MTT on different protocols. Any preference? what concentrations for each? Time of incubation?

Thanks in advance!!!

-wjchxl-

wjchxl on Apr 14 2010, 10:45 AM said:

1. you have to use Sea-plaque agar.
2. You can keep the agar at 65 degree C water bath to stop it from solidifying. You can just pour it and keep it back in the water bath. Then, you dont need to reheat every time.
3. Better to keep it inside the hood for 3-4 hrs. Let it solidify and transfer it to the 37 incubator. You can keep the lid of the plate open while solidifying the top layer.
4. Crystal violet is good enough to see. May be for 30 mins. but just a few drops of crystal violet. otherwise, you will get a violet background.

-blue_lotus-