Hi, this protocol works pretty well for my cells. But I'm wondering...

Hello FengZhi
Could you explain me the 8th step-Before you mix agarose gel with medium and cells. You should get pipette, tips ready to use, and perform it as fast as possible.Why is it the last place I couldnt get this step well.Thanks in advance.

It is agarouse-LE.

I would like to know if you got dried or shrunk gel after 3 weeks. If it is, please check your incubator if there is a tray with autoclaved water to keep your incubator 95% humidity. I have one experience to share with you. My first result was very good. the colonies were formed in the soft agar. One early morning, mo boss came and saw the results. He was excited and happy. At the same time, he assumed that the top of gel would become dry two days later( just be 3 weeks). He added some media. When I came three hour later, I saw the colonies were swimming in the plate: the soft agar status had been damaged.

Remember the top of soft agar would be dry since the incubator is not a moisture environment. If you forgot to add water in your incubator, it will happen. Under this situation, you may need add one or two drops of media to try to save your results. I do not have this experience.

Hello, your protocol doesn't mention the addition of media. After 2-3 weeks incubation, what if the top gel gets dry and shrunk?

Hi, Shane,
The purpose to stain the colonies is to count it easily and accurately, then take pictures for data documentation. Room temperature is fine. But 37C also is fine since the cells function well and can intake the dye fast. Thus, you should check it frequently. Once you find the agar also is stained, that means the time is too long. I stained it at room temperature. The time cannot be too long. I just watched it, once I found the agar became stained, I used absorbent paper to remove extra dye because the soft agar is fragile. I took pictures before and after staining. No staining is better than staining since the agar always is stained.
I do not think that preparing the crystal violet with ddwater is a good idea. The isotonic status to the colonies is better.