I am having difficulty in obtaining transformants, the positive control plasmid pKLAC2(malE) was prepared for transformation and only small colonies were observed. These did not secrete malE protein. I am worried that my competent cells are bad and I am reaching out for any tips, tricks and advice that you might have for me to determine if they are good or not. I will try to summarize my concerns, observations and questions. Q1: Are transformants on YCB (yeast carbon base) + acetamide (sole nitrogen source) expected to grow similarly to K. lactis on rich media? Even when I plate a very small volume of the transformation reaction, I get >300 small (0.1 mm) colonies after 24 hours. They proceed to grow slowly and I am aware that untransformed cells have weak acetamide degrading ability. I have subcultured a few select colonies and they grow fine on YCB + acetamide, albeit half as well as colonies growing on rich PDA media. They must not be transformants? Q2: Untransformed cells appear to be growing better than they should be....could it be possible that impurities in agar could be supplying nitrogen for growth? Small colonies after 24 hours can't be normal but I can't determine what is weakening the selection method. Or selection by acetamide is not as selective as I thought. Q3: After adding the transformation reagent to competent K. lactis cells, they remain in small (0.1 - 0.4 mm) visible clumps and aggregate. Is this normal? The solution never quite reaches a uniform turbidity, even after the incubation, heat shock and recovery steps. Q4: Unrelated question, galactose wouldn't go bad would it? I have a bottle from at least 30 years ago. I know that glucose has an indefinite shelf life (pretty much) but I'm not certain if "bad" galactose would explain the absence of protein expression.