pGLO Lab

The purpose of this lab was to perform the procedure of genetic transformation.  In this procedure we will be transforming bacteria with a gene that codes for Green Fluorescent Protein (GFP), which as a result will glow green under a black light.

pGLO plasmid is a plasmid that is used in making genetically modified organisms. The plasmid glows green because of the many reporter genes. The gene for GFP, which is encoded by pGLO plasmid, can be switched on in transformed cells when some sort of energy source is added to the cell. Cells that are transcribed and remain white if they do not contain arabinose, which is like a food to the cell, and cells that appear to be a fluorescent green are cells that have the sugar added to them.

First we labeled two separate micro test tubes +pGLO and –pGL. Then we added 250ul of transformation solution to each tube, and then immediately placed then over ice. After that we scooped up a single colony of bacteria and mixed it into the solutions of the tubes labeled +pGLO and –pGLO. After we examine the pGLO DNA solution under the UV lamp, we mixed a new sterile loop in the the pGLO plasmid DNA stock tube. We scraped a loop full of plasmid DNA and mixed it with the +pGLO test tube and NOT the –pGLO test tube. After that we let the test tubes sit on ice for ten minutes. In the meantime, we labeled our four agar plates, LB/amp +pGLO,  LB/amp/ara +pGLO,  LB/amp –pGLO, LB –pGLO. After the ten minutes was up we kept out test tubes in the sponge holder and placed them in a hot bath for 50 seconds.  Immidietly, we out the test tubes back onto ice for another two minutes after the 50 second hot bath. After the two minutes we added 250ul of LB broth to each test tube to act as food to keep the bacteria alive and to help them recover from the various temperature shocks. After letting the bacteria incubate in room temperature for ten minutes, we placed 100ul of +pGLO bacteria into the LB/amp +pGLO and LB/amp/ara +pGLO dish and the –pGLO in the the LB/amp –pGLO and LBn-pGLO dish. We then evenly smeared the bacteria around the dish and then closed them and stacked them upside down and placed them in the incubator.

We put the CaC12 in the bacteria in order to allow DNA to enter.  By placing it in the bacteria, the positively charged CA2 of the CaC12 will cancel with the negative charge of DNA allowing it to pass through the cell membrane of the bacteria.  By keeping each test tube submerged in ice, the bacteria is able to maintain its shape while allowing the DNA to enter.  When we heated the tubes, we heat shocked the bacteria.  This makes the DNA enter the bacteria.  By heating it, we are expanding the area for the DNA to enter.   After putting the tubes back in ice, this allows the gaps to close, keeping the DNA inside.  This process has created a new pGLO plasmid.  The broth is then used as food for the bacteria and allows it to create new proteins that are amp resistant.  By making it amp resistant, the amp  can no longer destroy the bacteria.  The bacteria is able to create more of the pGLO plasmid which in part glows in the dark.  

After analyzing our data, we are sorry to report that none of our plates glowed in the dark.  This could in part be because we failed to wait a certain amount of time after adding the broth.  Also, perhaps we had not successfully heat shocked our tubes as we were unsure if our tubes had successfully touched the hot water. However, we can safely conclude that when 

E. coli glowing in the dark after put under a UV light (glowing E. coli credits of lab group 6)

Gel Electrophoresis Lab

The purpose of this experiment was to figure out the location and amount of cut marks that each restriction enzyme had compares to the next

The point of this lab is to determine who or what unidentified DNA belongs to. The three restriction enzymes, in this case are PstI, Hpal and SspI are set up on three different columns. Once the enzymes are loaded into the gels the enzymes undergo gel electrophoresis. This procedure makes the enzymes separate into their cuts. This allows a scientist to evaluate who or what the DNA belongs to based on the cut marks.

To begin this experiment we poured about 5mm of agarose solution into a casting tray. Then we scooped out a large bubble of debris and added to the side of the tray while it is still a liquid. Once the agarose has set, we placed the tray in the gel box so that the slots are at the negative end. Once the slots for the DNA are submerged completely, the DNA is ready to be loaded. We then carefully extracted small amounts of the DNA out of the tubes, and then steadily inserted them into the chambers of the gel. It is very important that the gel does not break at any time, for the experiment will be ruined. Once they are loaded, the electrophoresis box is closed and connected to electrical leads. After some time, due to the shocks from the voltage source, the DNA begins to move along the gel, splitting at certain points. After the DNA has split down the entire gel, we took it out and examined the DNA cuts and determine what the DNA belongs to.

After examining our gel, we noticed that the DNA had travelled to a different area than where we had initially put it.  The DNA of each moved toward the positive end of the gel.  This is because DNA is naturally negative due to the phosphate backbone so it wants to move opposite from the negative end of the gel and is attracted to the positive end.  Also, the distance travelled by each strand of DNA was different as well. This is in part due to the size of the strands.  Bigger strands of DNA tend to not move as much as the smaller ones due to the fact it is harder for something bigger to travel a long distance.  The bigger strands are unable to move through the gel as easily as the smaller ones; in a sense, they can't fit.  They were the ones closer to the wells or the initial positioning of the DNA.

Our experiment allows us to conclude about the effectiveness of gel electrophoresis when looking for a DNA match. Each band moves a different distance because restriction enzymes only cut at their specific protein recognition sites. 

The gels after the DNA had been added, inside the machine having electricity run through it

Finished gel after having been shocked