My Favorite Project: Molecular Cloning I Blog #3

Over the summer, I have been given many projects in order for me to gain greater experience in the field of biology. However, one of my favorites has definitely been using molecular cloning. The first thing for molecular cloning is usually getting primers to PCR amplify a segment of DNA that you want to insert into a vector of choice.

Most people would usually view failure as an annoyance. However, failure is exactly what made this project my favorite. When trying to amplify a region of DNA with a thermocycler (device used for PCR), the results always failed. My mentor and I looked over the primers as well as viewed the protocol to see if there were any obvious errors. However, we could never find anything wrong with what we were doing. We therefore tried changing conditions numerous times in order for the PCR to work. Below is the image of most of our PCR Results. You can see two lanes with white bands. Those are just standard DNA lanes. There is no bands in the middle where we need them to be.

After many attempts, I was able to finally get the proper PCR results as you can see below. It was really rewarding to get the product that we wanted. This portrayed the saying “failure is the key to success!” Besides my favorite project, one of the coolest projects that I got to tackle this summer was with gel filtration chromatography. This is basically a method to separate proteins by size. There is a column with beads which contain pores. Bigger proteins will not fit inside the pores, so those proteins will elute first. Smaller proteins will move through the pores and go through the column. This will take the smaller proteins more time to go through the column and they will elute later. With this project, we wanted to run a known standard of globular proteins through the column and then run the fractions on a gel to see where the proteins eluted. This would give us standard we could follow to give an estimate on future projects with unknown protein size. Below is the awesome image of the gel that I ran. Proteins run on gels based on their size. Therefore, the higher bands are bigger proteins. In my opinion, the gel looks absolutely amazing. We can clearly see where the proteins eluted. 

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