My soft agar melt everytime when i do the soft assay, how to solve it??!!

1 post / 0 new
zhuangyanyan
zhuangyanyan's picture
My soft agar melt everytime when i do the soft assay, how to solve it??!!

Hi,all, thank you for reading my post and I really hope you can help me
I am doing soft agar assay for A549 cells, which is a kind of lung cancer cells. I use agar from affymetrix. It is called Agar(10907 100GM ultrapure) and there is no melting point has been shown in data sheet.
I use the protocal from Iruela-Arispe's lab from UCLA
For the bottom and top layer, I use 2x medium 2.5ml, FBS0.5m, 100X glutamine 0.05ml and 1.3% Noble Agar 2.5ml,
For the cell layer, I use 2x medium 1ml, 1x medium 2ml, FBS 0.5ml, 100X glutamine 0.04ml, and 1.3% Noble Agar 1ml.

So roughly, the concentration for soft agar in cell layer is 0.28% and in top and bottom layer is 0.58%. So after the soft agar plate is completed, I will add 2ml of complete medium on top of it every other day.

However, after 30 days, my plates always melt badly. Why is it turn out to be that??? And how should I improve it???

Hope you can give me some advises.Thank you