Previously I discussed how I am now changing an amino acid in the TEV cleavage site. (TEV stands for tobacco etch virus; it's the virus encoding this specific sequence) I am specifically changing the amino acid glycine to serine so that the TEV protease can cleave off the sequence more efficiently. In order to do this I had to amplify and isolate a plasmid containing the TEV cleavage site with the glycine amino acid; this would serve as the backbone of a recombinant molecule I am trying to create. I would then cut out the TEV cleavage site with the glycine amino acid and replace that with a similar sequence with the exception of the serine amino acid change.
Last week I first streaked a bacterial strain on a plate containing the plasmid backbone I needed for the cloning procedure. (This is the plasmid with the glycine amino acid in the TEV cleavage site)I allowed the bacterial colonies to grow overnight at 37 degrees Celsius. The next day I isolated a single colony and allowed it to grow up in LB media overnight. I then performed a mini prep (the technique I talked about in my previous posts) to isolate specifically my plasmid of interest.
At the same time I also performed PCR (Polymerase Chain Reaction; a technique used to amplify DNA) on the TEV cleavage site with the serine amino acid in place of the glycine. To confirm that the PCR worked, I ran the PCR samples on a gel to visualize the DNA bands. My PCR did work because in the gel image, the negative controls I had did not show while my samples did. I then performed a PCR cleanup to once again make sure I only have the DNA needed and not any other extraneous things. I obtained the concentration of my PCR samples with a nano drop.
After amplifying and isolating both the plasmid backbone and the PCR samples, I used the same restriction enzymes to digest or cut both the plasmid and the PCR samples. (restriction enzymes can be thought of as scissors used to cut DNA) By using the same restriction enzymes for the plasmid and PCR samples, I was able to create the same type of cuts which could ligate or bind to one another.
Today I ran the digested plasmid vector on a gel to separate basically the large backbone which I want from the small TEV site with a glycine amino acid which I don't want. I then cut out the gel piece with the plasmid and tomorrow I will be purifying it. After purification I can then ligate (or "glue together") digested purified PCR product and digested purified backbone.
Last week I first streaked a bacterial strain on a plate containing the plasmid backbone I needed for the cloning procedure. (This is the plasmid with the glycine amino acid in the TEV cleavage site)I allowed the bacterial colonies to grow overnight at 37 degrees Celsius. The next day I isolated a single colony and allowed it to grow up in LB media overnight. I then performed a mini prep (the technique I talked about in my previous posts) to isolate specifically my plasmid of interest.
At the same time I also performed PCR (Polymerase Chain Reaction; a technique used to amplify DNA) on the TEV cleavage site with the serine amino acid in place of the glycine. To confirm that the PCR worked, I ran the PCR samples on a gel to visualize the DNA bands. My PCR did work because in the gel image, the negative controls I had did not show while my samples did. I then performed a PCR cleanup to once again make sure I only have the DNA needed and not any other extraneous things. I obtained the concentration of my PCR samples with a nano drop.
After amplifying and isolating both the plasmid backbone and the PCR samples, I used the same restriction enzymes to digest or cut both the plasmid and the PCR samples. (restriction enzymes can be thought of as scissors used to cut DNA) By using the same restriction enzymes for the plasmid and PCR samples, I was able to create the same type of cuts which could ligate or bind to one another.
Today I ran the digested plasmid vector on a gel to separate basically the large backbone which I want from the small TEV site with a glycine amino acid which I don't want. I then cut out the gel piece with the plasmid and tomorrow I will be purifying it. After purification I can then ligate (or "glue together") digested purified PCR product and digested purified backbone.
You are making excellent progress. Nice work, Mariam.
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