In my previous post I talked about how I cut the plasmid vector backbone and my PCR product with the same restriction enzymes. Last week, I then run the plasmid vector on an agarose gel to separate my vector backbone which was approximately 7,000 base pairs in size from the piece I did not want which was approximately 78 base pairs in size. Because of the large size difference, it was very easy to see the position my vector backbone was at and I extracted out the vector backbone by actually cutting it out with a blade. I then used the Gel extraction/PCR cleanup protocol I briefly described in previous posts to purify the plasmid DNA from extraneous salts and whatnot. At the same time I also purified my digested PCR product. After this was completed, I ligated the PCR product with the change in amino acid to the plasmid vector backbone.
I then used electroporation to introduce the ligated DNA molecule containing the amino acid of interest into bacterial cells that can proliferate and make clones of my plasmid. Electroporators are basically that can generate an electric field in order to make bacterial cells permeable to the plasmid I am trying to insert into it. In this way I was able to transform bacteria so that it could take up the ligated DNA plasmid I had. I then heated the mixture containing the transformed bacterial cells and streaked these cells on a plate. I let colonies of bacterial cells to grow overnight in a 37 degrees incubator.
At the same time, I also used electroporation to introduce solely the plasmid vector backbone I had that did not contain my insert into bacterial cells. This was done to serve as a negative control. I streaked out these bacterial cells on a different plate to see whether for some reason I would see bacterial colonies grow. If they did, it would mean, I did something wrong and the plasmid ligated to itself instead of my insert. The plasmid was not supposed to ligate to itself because it was cut with two different restriction enzymes and making the ends of my plasmid incompatible to base pair with one another.
The following image shows the bacterial colonies I saw the next day.
Each small white circular dot is a bacterial colony. The plate on the left had the plasmid vector without the insert whereas the plate on the left had the ligated plasmid and PCR insert. There was one bacterial colony on the left plate, and over 30 bacterial colonies on the right plate. My mentor had said that 30:1 is a pretty high efficiency so basically the cloning seems to be successful.
This week however, I am screening for colonies, meaning I am picking 4 random colonies from the plate on the right to confirm that these colonies actually have the insert I am studying with the amino acid change. I will discuss more about the screening process in the next post.
I then used electroporation to introduce the ligated DNA molecule containing the amino acid of interest into bacterial cells that can proliferate and make clones of my plasmid. Electroporators are basically that can generate an electric field in order to make bacterial cells permeable to the plasmid I am trying to insert into it. In this way I was able to transform bacteria so that it could take up the ligated DNA plasmid I had. I then heated the mixture containing the transformed bacterial cells and streaked these cells on a plate. I let colonies of bacterial cells to grow overnight in a 37 degrees incubator.
At the same time, I also used electroporation to introduce solely the plasmid vector backbone I had that did not contain my insert into bacterial cells. This was done to serve as a negative control. I streaked out these bacterial cells on a different plate to see whether for some reason I would see bacterial colonies grow. If they did, it would mean, I did something wrong and the plasmid ligated to itself instead of my insert. The plasmid was not supposed to ligate to itself because it was cut with two different restriction enzymes and making the ends of my plasmid incompatible to base pair with one another.
The following image shows the bacterial colonies I saw the next day.
This week however, I am screening for colonies, meaning I am picking 4 random colonies from the plate on the right to confirm that these colonies actually have the insert I am studying with the amino acid change. I will discuss more about the screening process in the next post.
Hello Mariam,
ReplyDeleteI am really excited by your progress. So will you purify plasmid from the plate on the right and look at the restriction pattern on a gel? Also, in the old days we used to sequence the fragment to confirm the fragment and the junctions. Is that still done? Or do you use a different method?
Yes I picked four colonies and purified their plasmids yesterday. I was going to look at the restriction pattern on a gel, however because my plasmid is relatively large it is difficult to differentiate between linear plasmid DNA and supercoiled DNA. Instead, today I submitted the plasmid DNA I had to a sequencing lab which would sequence my fragment to confirm it.
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