Week 5 was my most independent, busy, and exhilarating week at the lab. With my PCR last week working, I was excited to move forward with the project and found myself thinking about my results 24/7. The next steps of my project this week included a restriction enzyme digest, alkaline phosphate treatment, ligation, transformation, colony PCR and mini prep, and a verification sequence. Needless to say I was busy! So in order to clone my CD 21 insert into a vector, I needed to cut a vector. I used an online program that visually displayed the vector, and I chose a site that would allow me to open it up and insert my plasmid. The vector I used was an pMND IRES-GFP vector and I cut it with EcoR1. Prior to the digest I had to do an alkaline phosphatase treatment, so that the vector wouldn't connect back together after it was cut. If my vector formed back together, my insert would never get inside! This process was new to me, but I followed an online protocol and completed it correctly. My RE gel picture displayed desired results, that I verified with the computer program.
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| RE results |
The next step was putting my insert into the vector. This process is called a ligation. Proper protocol is to ligate vector to plasmid in a (3:1) ratio. I placed 20 nanograms of insert into a 60 nanogram vector. This part of the procedure was complicated because it took a lot of equating and math skills. In fact, after I did all of this math, Dr. Mason showed me a much easier computer program to figure it out for me. She also laughed at me. After the ligation I had to transform bacteria to have this gene and vector. I had to make a happy growing environment for the bacteria, so I made LB + Ampicillin plates. I have used these plates before but never made them, so this was fun. Look how nice they are!!
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| my lovely bacteria homes |
I used highly competent cells to transduce the bacteria. These cells are basically easily manipulated in other words. So I put the vector with the insert into these cells and then grew them overnight on these plates. Spreading the cell and agar mix on the plates was fun because I used a plate spinner to spread it evenly. (I had a good time haha). The next day I was anxious to look at my plates, but they GREW YAY.
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| colonies |
The next step was to see if these colonies in fact were transduced. To do this I did a colony PCR of random colonies (20 of them).
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| here are some bacterial samples and tubes for a miniprep I completed |
I picked a primer that would amplify a specific 1200 bp fragment and thus show correct orientation if the colony had the insert. A few of the colonies did have the insert and showed up on the gel. Here is one for example:
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| this bacterial colony was transduced!!! |
I then sent the colony minipreps to sequencing that had the DNA band. It was verified that my bacteria were in fact transduced! I WAS EXHILIRATED!! I regrew this same colony on a seperate plate and made a glycerol stock for future use. The next step of this project is to complete a MAXI PREP of the bacteria to amplify these transduced cells to later use for T cell redirected therapy. I will be doing maxi preps in the lab during week 6, and Dr. P is visiting !! Woo hoo.
I also did some clinical trial data stuff for Dr. Mason that is important for getting the vaccine pushed through for children with pediatric osteosarcoma. Each dog on the trial has a folder, in which I was entering information into data sheets and adding medical files to them. I organized them nicely for her.
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| greetings from the Mason Lab |
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