We transformed yeast with lacZ and the experiment worked. We cryopreserved the yeast and now I'm not getting any beta-gal activity.
I didn't add the DTT to the buffer with the ONPG, but did add DTT to the buffer used to lyse the cells. Everything else was followed by the book.
Could this affect the results that much? Or did my yeast lose their plasmid?
You definitely need DTT in the buffer otherwise the assay will not work, so not sure you will have enough present for the reaction if you only added it to the lysis buffer. That would be the first think I would try. It could also be that you just lost activity. But if you are using a positive control then you should be able to distinguish these.
Hi there,
As mentioned above, you can't state on the assay (DTT requirement) unless you have run this assay with a positive control.
BTW why do you suspect a plasmid loss?
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