Cloning dilemma

For once, I wanted to distance myself from everything that happened inside those four walls — the cloning experiments that failed, the endless PCR troubleshooting, and the electrotransformation that didn’t turn out the way I hoped. 

I told myself, this one week-break was supposed to be a time to retrace which protocols I overlooked.

 

Double digestion? Done 

Gel-purification? Done 

Ligation mix? Done 

Electrotransformation? Done 

Recovery of transformed cells? Done 

After 16-18 hours of incubation at 37°C on the selective antibiotic agar plates, I saw…there were colonies. I mean, TRANSFORMED COLONIES…

For a brief moment, I genuinely let myself believe it had finally worked. 

But, science has a way of humbling you. 

I picked the transformed colonies, already anticipating the next steps. Colony PCR first. Restriction digestion after that. 

Then… reality hit. 

No expected PCR band. No plasmid to verify… The colonies were there, but the plasmid wasn’t. I still confused, trying to make sense of these endless questions. 

What went wrong? Did I miss something? 

I need answers! — so, I did what every clueless postgraduate student does — I reported (ok… maybe I literally cried) to my supervisor. After all, he's the only premium researcher adult — An expert sifu who's practically capable of turning these countless failed cloning experiments in the lab into an instant diagnosis of what went wrong. 

“It’s probably AMR. Antimicrobial resistance” 

I blinked. I was fully prepared for a Nobel Prize-level explanation. Instead, I got three letters: AMR… 

“What antibiotics are you using?” he asked. 

“Kanamycin,” I replied. 

“OK… Shewanella is resistant to kanamycin”. 

Oh… that was it? I wasn’t convinced because I was expecting something more complicated — a failed ligation, inefficient electrotransformation, or some hidden experimental mysteries that only an expert researcher adult could decode. 

Then, came the final verdict of this cloning dilemma. 

“Get a new kanamycin from our supplier,” he said. “And get a ligase that works at room temperature. Your ligation most probably failed”. 

Honestly, I couldn’t agree more… I’ll work on it next week.