Emily Guo, Week 4, Successful Plasmid Transfection!

Anna and I have spent this week trying to create the plasmid for our second gene. After annealing our oligos and doing the ligation reactions, we transfected and plated our E. coli cells on Wednesday and hoped for colonies Thursday morning. And we got them! Only our negative control plates were empty of colonies, and the rest had a much higher yield compared to the transfection we did for the first gene. As expected, less ligation reaction (3uL) and a 1:200 insert dilution seemed to yield the most colonies. I showed Dr. Peretz the results when she came to visit that day.

Colonies (on our positive control)

For lunch, Dr. Peretz, Scott and I went to eat at a nearby Dallas BBQ, where everything is truly Texas-size.

On Friday, we extracted and purified the plasmid DNA from four of our samples (the colonies of which we had picked on Thursday). After we checked the quality and quantity of our DNA on the Nanodrop machine (a value of 1.8 - 1.9 indicates good quality for DNA, 2.00 for RNA), we prepped 1ug of DNA from each sample for restriction digestion, which we will run on Monday. Once again, we will be using the restriction enzymes AflIII and XbaI, which we confirmed via NEB. Because the gel needs at least 5 hours to run in order to maximize the difference in distance traveled by the short pieces of the cut plasmid, we cannot run the gel in one afternoon, so I will be making the gel (2.5% Metaphor agarose) first thing Monday morning.

Unfortunately, the qPCR we ran on Monday flagged the samples ran with primers for three of the six genes (for which we wanted to assess gene expression). One problem was multiple peaks in the melting curve, which indicated that two sets of primers were off target and needed redesigning. Furthermore, samples ran with two sets of primers had CT values that were too high (in the mid 30s), indicating that the quantity of cDNA was too low for those primers. A CT value is the number of cycles needed for the fluorescent signal of a sample to cross the threshold during PCR. Because higher CT values indicate that a sample needed to go through more PCR cycles, they have a higher chance of being less accurate. That wouldn't be too much of a problem, but because CT values were a bit on the high side for all of the samples (not just the flagged ones), this meant that we would have to run tests to determine the optimal cDNA dilution to use across all the primers. It will take some time to do these tests in addition to ordering the primers, so the next time we will run qPCR would most probably be next week.

I designed a set of qPCR  primers using Ensembl, Primer 3 and BLAT 

All the while I’ve been helping Alyssa with cell culture. In addition to the quick "aspirate and add media" care done every few days, I've also been passaging the cells when they became too overcrowded. Passaging adherent cells is a very time consuming process as Trypsin is needed to hydrolyze the proteins that allow the cells to attach to the bottom of the wells, and then the cells need to be centrifuged down and re-suspended with fresh media before being pipetted into new wells. Suspension cells are somewhat easier to passage as they are already suspended in growth medium. I'm taking great care to avoid contamination across wells, and I'm grateful for their continued trust in me.

Adherent & suspension cells; the pink growth medium yellows, indicating a change in pH and  overcrowding of cells. If fresh media begins to change color very soon after it is added, it is time for passaging.