Week 6 at the Deo Lab-- Zui Dighe

This week the other lab technician who is on the same project went for vacation, so I was all alone. This gave me even more independence to come, work, and go as I pleased. It took a bit longer to find things but I was able to continue along.

Another problem occurred this week. The competent bacterial cells used for transformation turned out to be incompetent. Thus, I had to make competent cells. I printed out a protocol with the help of my PI and began the process on Tuesday. The process took the whole week and I will test them out on Tuesday to see if they work. If the homemade competent cells are successful, I will help save the lab thousands of dollars. 

To make competent cells first I had to combine lots of chemicals and calculate correct dilution numbers. This is for LB, magnesium chloride, calcium chloride, and glycerol. No one in the building seemed to have plain LB plates (without antibiotic) so I had to make those as well! This took a day and a couple hours for cooling. Next, I used a zigzag method with a pipet tip to streak LB plates with the Top 10 cells that did not work. I would be reactivating the cells. 

I made three LB plates and let them grow overnight. I was looking for single colonies on the plates. That is what streaking is for (to prevent multiple colonies). Out of my three plates, two had single colonies. Yay, good results. I picked the colonies with a pipet and added them to a 250 mL LB mix in a beaker. The cells would cultivate overnight. 

The next day I measured the OD of the cells using a cuvette machine. OD measures the amount of bacterial growth. For competent cells I could not let the OD grow over .9. Thus, I had to keep running upstairs and measuring to keep the cells in check. 

On Monday I will perform a bunch of centrifuging, decanting supernatants, and re-suspending with new chemicals to reactivate cells. Then I will flash freeze the competent cells in a mixture of ethanol and dry ice. 

Above is a video that I took in our microscope room. It is of zebra-fish embryos from the other project in my lab. The spine-like structure is the forming artery wrapped around the fish's veins. The color is green because of gfps (green fluorescent proteins).The other lab is classifying heart disease mutations in zebra-fish to further understand the process in humans.