Harvesting pDNA

Bhuvi M -

Hello everyone! Last week, I initiated a new transfection for Flo with MAL and OE33 with Axl. I quickly realized that we did not have enough Axl pDNA.

Thus, we needed to harvest more pDNA. Plasmid DNA, typically abbreviated as pDNA, refers to small, circular DNA molecules found in bacteria and other microorganisms. They can replicate independently of chromosomal DNA and often carry non-essential genes for the organism’s survival.

To replenish our Axl pDNA stock, we utilized bacteria stocks containing the Axl pDNA from the -80 degrees Celsius freezer to streak agar plates treated with ampicillin. The presence of ampicillin prevents contamination as the bacteria are resistant to it. Additionally, we took precautions to work near an open flame to minimize contamination risks.

The following day, we selected isolated colonies using a pipet tip. We transferred them into 6 mL tubes containing LB Broth and another antibiotic, such as ampicillin, to ensure only bacteria with the desired gene could grow. These tubes were placed on a shaker overnight at 200 rpm at 37 degrees Celsius, optimal conditions for bacterial growth.

Plates streaked with bacteria.
LB Broth with Ampicillin and pipet tip with bacterial colony.
Tubes with LB Broth, Antibiotic, and Bacterial Colonies on the shaker overnight.

The subsequent day, the lab technician isolated the pDNA using the QIAamp DNA Mini Kit.

Next week, we plan to proceed with transfecting the newly harvested pDNA. Additionally, we aim to commence inhibition studies.

Furthermore, I had the opportunity to visit TGen last week, where I gained insights into data analysis techniques commonly employed in genetics.

Thank you for reading!

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    Hi Bhuvi, great work and good luck with the inhibition studies next week! Also, what were some of the data analysis techniques used in genetics that you learned at TGen?
    I learned primarily about spatial transcriptomics, which is the study of all RNA molecules in a cell, including their expression levels, function, structure, and regulation. Additionally, the machine, GeoMx Digital Spatial Profiler, can give a specific x,y coordinate to the RNA molecule to classify a cell, and allows us to further understand what types of cells drive diseases like lung cancer.

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