For weeks, I struggled to produce a successful harvest of lentivirus. I needed to transduce my pancreatic cancer cell lines with shRNA targeting my gene of interest. This is a common protocol in my lab, and I had never experienced difficulty accomplishing this task before. Now, I was unlucky beyond all reason. My plasmids were the correct sequence and intact. My transfection reagents were brand new. My packaging cells were boastfully glowing red with the mCherry from my cDNA transfection control. Yet, once harvested, no virus would infect my target cells, and my experiments were aggravatingly halted. With the despair only known to the senior graduate students living with that chasm of unfathomable emptiness in their innards, where that terrible voice whisper-shouting, “YOU’LL NEVER GRADUATE,” echoes brutally, I attempted my protocol once more. I stared hopelessly at the box of Whatman Puradisc PES 25mm, 0.20 micron syringe filters and… wait, what? 0.20 micron? These are supposed to be 0.45 micron. Are you kidding me?! For weeks I had harvested viral supernatant with a filter that had pores too small. I was extracting the virus right out of my supernatant, never to reach my target cells. Curse you, Whatman Puradisc PES 25mm, 0.20 micron syringe filters. Curse you.
PEER EDITED BY Chris Givens AND Erinn brigham
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This article was co-published on the TIBBS Bioscience Blog.