Science Fail Monday: Cloning CarelessnessPosted: 06 29, 2015
One of my research projects involves characterizing a new protein-protein interaction. I wanted to know which specific regions of the two proteins interacted with each other, so I generated plasmid DNA able to express several shortened versions of the proteins in cells, hoping to later test whether the proteins could still bind to one another. Cloning plasmids is supposed to be an easy job for a molecular biologist, and the steps are fairly straightforward.
I made copies of the portions of DNA that I needed and added restriction endonuclease sites so that I could fit the fragments into the expression vector. I cleaved the DNA products, placed them in the expression vector and transformed the new vectors into bacteria. The result? Plenty of colonies. Everything was working flawlessly on the first try, which never happens to me while cloning. I was ecstatic. To confirm my results, I sequenced the plasmids and they all looked perfect except for one thing:
I had forgotten to add a start codon to the DNA, which is the equivalent of making a rubber balloon and forgetting to add a hole through which to blow air. And, the vector did not have one of its own, meaning that my flawless start was merely a hoax: neither mRNA nor protein would actually be made from my plasmid.
Crestfallen when I realized that I still had never gotten cloning 100% right, I made myself think the only positive thought that I could: at least I had not wasted even more time wondering why my proteins were not expressing.
Written by: Nicole Carlson
Illustrated by: Bailey Peck
Peer edited and reviewed by Rachel Dee & Mejs Hasan
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This article was co-published on the SWAC Blog, The Pipettepen.