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Submitted by Mandy Hunter on Fri, 10/11/2013 - 7:32pm
Who is the microscopic answer to the Blue Man group? Find out in this week's blog post!
Well, they sure weren’t cute little carrot colonies.
There was a serious dichotomy to this week’s work. Everything either turned out fabulously or fell flat on its face. Let’s start with Cell Culturing:
The carrots look like an entirely successful experiment in growing mold and bacteria, so it was back to the drawing board on that one. And I’m seriously mourning my lovingly-tended CHO cells. I have a feeling that next week I’ll be doing the Chinese hamster ovary equivalent of flushing good bye during a gold fish burial at sea. After babying those cells, speaking softly and sweetly, playing light jazz (not really) and subculturing oh-so-gently, I betrayed them by attempting a calcium phosphate transfection and they were not happy about it.
First, we made two separate solutions. Solution A was 36 μL of 2M CaCl2, 20μg of the DNA containing that lovely fluorescent gene I was telling you about, and 300 μL DI H2O to bring it up to volume. Solution B was 300 μL 2X Hepes Buffered Saline (HBS). We added Solution A dropwise to Solution B while bubbling air through the mixture with another pipette and I think this is where it all went wrong. My solution was blue and I didn’t realize until afterward that everyone else’s solution was clear. After investigation, I noticed that the blue filter in my pipette tip was dislodged and hanging out at the bottom of the tip, which means that the blue color I assumed was the result of a chemical reaction was just dye from the filter. Sigh.
Anyways, my blue solution was incubated at room temperature for 30 minutes and then dribbled all over my CHO cells. I’m sure the poor little things recoiled in horror when they realized I was inviting the microscopic world’s answer to the Blue Man Group to come take up residence in their homes. We then incubated them overnight at 37°C in a humidified CO2 incubator. When we checked out our handy work on the FLoid, here’s what mine looked like:
Here’s what it should have looked like:
Thankfully, my classmates are generous when it comes to sharing results.
The kicker is that we selected for the cells harboring the fluorescence by introducing Gentamicin (G418), so even though my instructor assured me that after the weekend I may be pleasantly surprised by the survival of some of my cells, I’m thinking I’m gonna be bugling TAPS for the little gals.
But perhaps their deaths were not in vain. In Mo Bio my competent E. coli JM109 cells evidently witnessed the carnage wrought across the hall in Cell Culturing and figured out quickly which side their bread was buttered on, because when I added 10 μL of 0.005 μg/μL pAMP plasmid DNA, they took it up BEAUTIFULLY and promptly began growing on LB AMP agar.
Guess they knew what was good for them.
In other news, I am seriously stoked to say I’ve been afforded the opportunity to meet some of my Bio-Link buddies at the ATE conference in October (Dr. Sandra Porter and Jennifer Newsted, we seriously need to get a coffee together).