Week 8: More Validation

This week, I began to restart the entire process again to acquire more material for further validation.

Inoculation

To prepare for a midi prep, I took my three samples from last week’s transformation and started inoculation. I grabbed 3 new falcon tubes and labeled them GFP, WT, and N-mut. As Steph had pre-prepared liquid LB with kanamycin (the antibiotic that the GFP construct is resistant to), I added 35mL of this solution into the GFP falcon.

To make liquid LB with the ampicillin antibiotic, I added 35mL LB broth to the WT and N-mut falcons. To add the ampicillin antibiotic in a 1:1000 dilution, I added 35µL to each falcon.

Next, I opened the LB agar plates one at a time and inoculated a single colony from each using a pipette tip. I dropped each tip into its respective falcon and swirled the falcon before pouring its contents into a new 250mL beaker. After covering each beaker with aluminum foil, I left them in the shaking incubator overnight.

Plasmid Midi Prep

The next day, I took my flasks from the incubator, and they were cloudy – a sign that there has been bacterial growth! I then conducted a midi prep to extract and purify my amplified plasmids. When I was finished, I left them in the -20˚C freezer for later use.

Contamination (!)

When I checked on my HEK293T cell confluency (see last week’s blog), I discovered an unfortunate situation — my cells were contaminated! Sadly, we had to bleach the cells and dispose of them. 

Hopefully, we’ll get a chance to split more healthy cells next week when Steph’s HEK293T cells are confluent enough for more splitting.

More Validation

For the rest of the week, I did some more validation. As our cathepsin A antibody finally arrived, I added it to the membrane I’ve been using for the past few weeks (the one with both SM and IP). I obtained the image below:

That’s it for this week. Hope to see you all again next week!