I accidently found the key to Pir1 cells in frustration. We optimized the zeocin dose and got no colonies. I was so frustrated I didn't even bother to throw them away. I just shoved them back into the incubator and took two days off and then the weekend came. That Monday when I came in to throw out the plates, I had big fat colonies! I ran a mini after I streaked a new plate for each colony picked and got no DNA. I was again frustrated. But the next day, I ran another mini on the newly streaked plate( which the colonies grew over night), and got DNA! So from now on I leave the plates for two day instead of one. You can see teenie tiny colonies on day one, but they are very robust on day two. This goes against all cloning procedures but it works.
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