Next Step: Transcription Study

I've been working hard to build the plasmids (self-contained pieces of circular DNA) that I'll need for my experiment on DNA transcription (how DNA is read). After the gBlocks and oligos that I ordered came in, the next step was to make the pUC19 backbone that I would then be able to clone the Broccoli and Spinach gBlocks into.

In order to do this I ran the pUC19 plasmid through a PCR with two oligos (short pieces of DNA that allow me to make copies of the larger pUC19 template). The initial results of this procedure were inconclusive. I repeated this procedure another three times, taking a total of three extra weeks, until we found the problem to be an issue with the program on the PCR machine, which I successfully fixed.

After preparing the pUC19 vector, I then used a Gibson cloning reaction to insert the Spinach and Broccoli sequences into the plasmid.

I grew up these cultures on Luria Broth agar plates and found numerous colonies for both Spinach and Broccoli variation plasmids. I then did a Mini Prep to isolate the plasmid DNA and then analyzed samples of the DNA that were cut with restriction enzymes to make sure the DNA was the expected size (it was!).

The next step will to be to send in the final product for sequencing and, if everything looks right, use this product to finish the transcription study segment of the experiment.

Thank you all so much for your patience.

Good science takes time to complete and since I'm learning as I go it is taking even a little bit longer. We have had some unforeseen delays and we are farther behind schedule than we would like, but we're getting things back on track.