Calcium Chloride Lab Report

This lab is about moving genes from one thing to another using plasmids. Plasmid has the ability to replicate, so it replicates independently, and separately from the chromosomal DNA. Plasmid are one or more small piece of DNA and they enter cells as a double strand DNA. When they enter the cell as a doubke strand they do not invade he chromosomal DNA. We will also transform bacteria into GFP which is mainly from the jelly fish Aequorea Victoria. The GFP causes the the jelly fish to fluorescent and glow in the dark. After the transformation, bacteria starts to make the GFP which causes them to glow a green color under a ultraviolet light.

The pGLO plasmid is engineered to express green fluorescent protein (GFP) in the presence of the sugar arabinose as well as the ampicillin resistance gene β-lactamase (bla) (Brown, 2011). Original E. coli HB101 do not have ampicillin resistance or the GFP gene allowing them to glow under UV light. In this experiment, we transformed E. coli HB101 with the pGLO plasmid by heat shock to make the bacterial cells competent, allowing the plasmid to enter the cell (Brown, 2011). The mixture of bacteria with pGLO plasmid were given recovery time after heat shock, then spread on LB/amp and LB/amp/ara agar plates. The bacteria mixture with no plasmid added were spread on LB and LB/amp agar plates and all four plates were incubated at 37°C for

The purpose of the PGLO lab was to be able to perform a procedure known as a genetic transformation. We used a procedure to transform bacteria with the gene that codes for a Green Fluorescent Protein (GFP). The actual source of the GFP gene that we used in this complicated experiment is the bioluminescent jellyfish Aequorea victoria. This protein causes the jellyfish to glow under a UV light that was provided in the dark. After the transformation procedure, the bacteria showed their newly acquired gene from a jellyfish and produced the fluorescent protein, which as a result, causes it to glow. If the bacteria glowed in the dark, that was the initial sign that the experiment was successful.

The experimental part of the lab consists of setting up the materials needed. A sample of E.coli and a solution of calcium chloride are first obtained and placed in different test tubes. 630µL of Calcium Chloride (CaCl2) are then removed from the test tube and inserted into the test tube containing E.coli cells (Alberte et al., 2012). The newly formed substance of Calcium Chloride and E.coli is then mixed and incubated in ice for 10 minutes, making the cells more competent. Two test tubes are obtained and labeled; the first test tube is labeled with pUC18 and the second one with “Lux” to represent the plasmids being used. These two test tubes are then incubated in ice. 3µl of the set plasmid are added to each of the two test tubes. The test tubes are tapped to guarantee the cells are well

In the pGLO Bacterial Transformation lab, Escherichia coli is transformed with a gene encoding green fluorescent protein by inserting a plasmid containing the GFP gene, beta-lactamase, and arabinose into the bacterium. Successfully transformed bacteria will grow in the presence of ampicillin and glow a bright green color under ultraviolet light. The sugar arabinose is responsible for switching on the GFP gene in the transformed cells, without it, the gene will not be expressed.

Spin the two tubes in a centrifuge for 5 minutes on opposite side of the centrifuge. The bacterium will collect at the bottom of the tube, so pour out the extraneous supinate. Then, add 250 microliters of buffer. The Ca2+ cation of the buffer neutralizes the repulsive negative charges of the phosphate backbone of the DNA and the phospholipids of the cell membrane allowing the DNA to pass through the cell wall and enter the cells. Place both tubes on ice. Then add 10 microliters of water into one tube and 10 microliters of plasmid DNA into another tube labeling the one with DNA with a + and the one with water -, and place on ice for 10 minutes.

How does the addition of pGLO plasmid to a solution containing E. coli bacteria affect the growth and characteristics of the bacteria? Genetic transformation is the incorporation of foreign DNA into an organism to potentially change the organism’s trait. Plasmids are small circular DNA that replicate separately from the bacterial chromosome. In nature, these plasmids can be transferred between bacteria allowing for the sharing of beneficial genes. Due to this characteristic, plasmids allow for genetic manipulation and can be moved between bacteria easily. The pGLO plasmid utilized in this experiment encodes the gene for Green Fluorescent Protein (GFP), which under the right conditions can produce a glow. The gene regulation system present in the pGLO plasmid requires

The color of food is an integral part of our culture and enjoyment of life. Who would deny the mouth-watering appeal of a deep-pink strawberry ice cream on a hot summer's day or a golden Thanksgiving turkey garnished with fresh green parsley?

The purpose of this lab was to find out how gaviscon reacted with different salt solutions. Sodium Alginate is a compound that forms with the Gaviscon and forms a “raft” and blocks all the stomach acid from traveling up the esophagus.

To do a transformation one micro test tube was labeled +pGLO and the other was labeled –pGLO, using a transfer pipette 250 µL of transformation solution was added to each tube in the foam rack. The tubes were then placed on ice for three minutes. During these three minutes, a sterile loop was used to pick up a single colony of E. Coli from the starter plate by gently running the loop over the agar. This loop was then inserted into the +pGLO tube and the loop was spun until the entire colony dispersed. Using a different sterile loop, the same procedure was used for the –pGLO tube. After both tubes had their own colony of E. Coli, they were placed on ice for another three minutes. DNA plasmid was added to the +pGLO tube by taking a new sterile loop and immersing it into the stock tube creating a film across the loop then inserting

The focus of this lab was to identify which plasmid (PFG or Pglo) was inserted into the E. coli culture. In order for the E. coli to be transformed, the E.coli must first be made competent. The cells were made competent by incubating the cells in calcium (2+) and then in cold temperatures. A quick heat shot opens pores in the cell membrane and allowed plasmid DNA to enter the cells. The plasmid that was inserted into the E. coli culture had antibiotic-reistance gene and a reporter gene. A reporter gene is a gene that is easy to detect, and for this lab it could be Pglo or PFG. The cells were placed in plates that contained antibiotics to kill any bacteria that did not uptake the plasmid, this is necciary because about 1% of bacteria will be transformed. This allowed only cells that up took the DNA to

While conducting this experiment many questions came to mind what effect can plasmids had on the transformation of E. coli and why did CaCl2 had to be used. During the experiment the solution was used to neutralized negative charges and heat sock was done to traumatize the cell membrane to get the cell ready for the intake of plasmids. As shown above observation can be made from the agar plate which describes the affect that plasmid had on the each. First, with the LB c, LB np and LB lux DNA exhibited lawn growth consequently lux brought about the bioluminescence effect on plates LB lux. This was the result of the absence of the Ampicillin antibiotic and the lux together. So, agar plates labeled LB/AMP c and LB/AMP lux, shown colonial growth which is a clear indication that minimal

In conclusion, using spectrometer to determine the absorbance spectrum of each test tube filled with different mL of Cobalt Chloride Hexahydrate. Using a weighing boat, 5.9485g of Cobalt Chloride Hexahydrate was dissolved in a 100mL of deionized water. When the first 0.25Ml was measured out with a graduated cylinder, to get 0.20ml, 0.15ml and 0.1ml. Dilution was repeated to fill up the solution in the volumetric flask up to 100

Then, we placed 100 µl of the E. coli cells into four Eppendorf tubes each and labelled them A, B, C and D. After filling them up, we placed them into an ice box. After this, we needed to perform the transformation process by mixing the E. coli cells with different concentrations of plasmid DNA. In test tube A, we added 5 µl of distilled water to the E. coli cells. In test tube B, we added 2.5 µl of undiluted plasmid DNA to the E. coli cells. In test tube C, we added 1.0 µl of undiluted plasmid DNA to the E. coli cells. Lastly, in test tube D, we added 1.0 µl of plasmid DNA diluted 1 in 10 to the E. coli cells. Then, we used a P100 pipette and mixed all the liquids in all the Eppendorf tubes by pipetting the solution up and down. Then, we left the Eppendorf tubes along with its contents in the ice box for twenty minutes. After twenty minutes, we labelled the base of the four agar plates to match with the appropriate plasmid DNA concentration about to be plated. We also wrote our initials, the date, and the table number on the base of the agar plate. We then gently lifted up the cover of the agar plate with just enough space to allow us to plate each tube of cells onto four different agar plates – one agar plate to one plasmid DNA concentration. We used a spreader to spread the cells evenly over the surface of the agar, being careful not let the spreader touch

6. The precipitate may have not dried up properly making it so there was water adding weight on it.