Were you using the correct concentration of hygromycin and did you cool the media before adding it.

Hmm, it could be a PCR issue. Colony PCR can be a bit tricky. What are your PCR controls?

It could also be a colony issue (not really resistant). I found out the hard way that while you can make and keep a stock of hygromycin in the freezer, freezing the original bottle from the company can lower the efficacy of the selection. In brief, when I was a student my PhD lab moved in the winter and some chemicals were damaged by being exposed to freezing temperatures in the moving truck.

Hi Rudrakshi Sharma

Colony PCR is a bit tricky. The denaturation step and the amount of colony are important.

Inoculate a very little colony in 5µl water and denature at 95°C for 10 min. Then add 25µl PCR mastermix, mix it well and run your PCR. Do not take agar media when you are inoculating the colony. The agar plate components may inhibit PCR. If the colony amount is high, then dilute it 10 folds before using it.

Sometimes normal PCR protocol does not work. You can try a step-up PCR protocol in this case.

Best wishes.

Yes, I followed all the steps as you all wrote but this time I am getting a mat-like growth on the hygromycin plate after plating. This is very unusual. One of the determinants for my mutant strain is that It can't grow at 37 degrees. I don't know why there is so much mat even if I am plating less. Any valuable input regarding this?

Hi Rudrakshi,

I have had similar problems recently so just wondering if your problem has been solved. I have done the yeast transformation for the gene integration (tagging a specific gene) using selectable marker geneticin. Similarly, I always got a law of cells after one or two overnights even though I spead less cells.

Besides, I got small colonies on the plate after 3 days. I did colony PCR and found only two out 25 seemed to be correct but I'm not 100% sure. Here's how I did it: I had a WT group (primers anneal to upstream and downstream of the stop codon of the specific gene so there should be no bands if the specific gene is tagged) and a Tagging group (primers anneal to the KanMX gene and the downstream of the specific gene's stop codon so there should be a band if the specific gene is tagged). However, now I got a band in both WT group and the Tag group. The size of the band in WT group seemed to be the size of the tag, but there should be no band. I included a positive control, the same gene tagged with the same tag and selective marker in another yeast strain. This control showed band in Tag group and no band in WT group.

Please provide your valuable suggestions for my yeast transformation and opinions on if that 2 out 25 colonies worked or not considering bands shown in both WT and Tag groups. Thank you.