Photosynthesis Lab Abstract

After it was expelled back into the cup, 1 ml of the saline rise was transferred into a micro test tube in order for it to be spun in a balanced centrifuge. The micro test tube that contained the 1 ml of saline rise was inside of the centrifuge for 2 minutes, resulting in cells at the bottom of the tube. Since all of the cells were present at the bottom of the tube, the saline was poured off and the tube was vortexed to be sure that there were no clumps of cells. Afterward, the InstaGene Master Mix (which removes cofactor to inhibit DNA cutting enzymes) was vortexed with the saline rise in order to fully mix the contents of the tube (Bio-Rad Laboratories, Chromosome 16: PV92 PCR). The tube was then incubated at 56 degrees Celsius for 10 minutes to inactivate DNAses and put on a second heat block at 100 degrees Celsius for 5 minutes to disrupt cell membranes. The tubes were put into a centrifuge then cooled down in a 4 degrees’ Celsius fridge.

Does carbon concentration affect the rate of photosynthesis? The carbon concentration does affect the rate of photosynthesis.

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.

The second method was to purify plasmid DNA from the E. coli culture and physcically analyze the DNA by digesting the plasmid with specific restriction endonucleases. The restriction endocucleases used were Bam HI (B), EcoRV (E), and BgIII (G). These restriction endonucleases recognize specific short DNA squences. These cut plasmids were then placed in a 1% aragrose gel to determine the band sizes. The expected phenotype for Pglo is that there will be floursenscens light produced on the LB + amp + Arab plate, where as for PFG the expected phenotype is that there will be flourcent phenotype only in LB+ Amp + IPIV. For the physical analysis Pglo will produce fragment sizes in restriction enzyme (E) 5371bp long. Enzyme (B). fragment sized 625bp, 220bp, and 4,526bp in restriction enzyme (B). Pfg will produce 2 fragments in restriction enzyme (E), one will be around 3,855bp long, and the other one will be 2,506bp long. In restriction enzyme (B) there will be only one band about 6361bp long. From restriction enzyme (G) there should be one bad around 6361bp

The technique that allows scientists to follow with their own eyes the dynamic movements of specific proteins as they occur within the living cell is the green fluorescent protein. This is a protein that is extracted from only a few jellyfish. This diffuses a green fluorescent colored light. The green fluorescent protein can be blended to the protein and can work normally and so does the protein itself that it is binded to. The protein is not affected and can be moved and transported throughout the cell and can then be seen.

Firstly, the lab was done within a period of 75 minutes. Time efficiency was a factor that could have been more accurate when creating competent cells. Competent cells are able to take in extra foreign DNA. The procedure involved timing for series of events. Events such as: both tubes on the ice for 10 minutes, both tubes in the bath for 50 seconds, both tubes on ice for 2 minutes and both tubes at room temperature for 10 minutes.

The purpose of the experiment was to isolate plasmid DNA, followed by restriction digestion using restriction endonucleases and then visualizing the digested fragments after subjecting to gel electrophoresis. Plasmid DNA (pSP72 DNA) was isolated from Escherichia coli KAM32 (E.coli) cultures using the QIA prep miniprep kit and then subjected to restriction digestion by EcoRI and HindIII. The restriction digested DNA was then loaded into the wells of 0.7% agarose gel and subjected to electrophoresis. It can be concluded from our results that our plasmid DNA isolation was successful and the restriction digestion results were partially in agreement with our hypothesis.

 DNA is in the living cell and it can be subject to many chemical alterations.

They were run at 1X 94ºC for 3 minutes, 30X at 94ºC for 30 seconds; 50ºC for 30 seconds; 72ºC for 45 second and 1X at 72ºC for 5 minutes. The PCR reactions took about 1 hour and 30 minutes to complete. The PCR products, were then purified by removing the leftover primers, nucleotides and salts. 250 µl of Buffer BB were added to Tube B and the mixture was pipetted into a spin column. The mixture was centrifuged for 30 seconds at room temperature. Then 2 cycles were completed at 30 seconds each with 200 µl of Buffer WB to remove any impurities. Then 25µl of Buffer EB were added to the tube to release the pure DNA and the mixture was centrifuged for 30 seconds. As the PCR reaction was running, a microscope slide was prepared from the live bacterial culture to observe the individual cells of the unknown bacteria and determine its

All of the T-streak showed growth however, not all of the bacteria formed colonies. Pseudomonas aeruginosa had no colonies therefore, the colony morphology were not applicable. The error could have been to not spreading the bacteria enough using the T-streak method. P.aeruginosa, Enterobacter aerogenes, Escherichia coli and, Klebsiella pneumoniae all tested as gram negative.

Photosynthesis and respiration are “reversible” because both are apart of energy flow and chemical recycling in the ecosystem. Light energy, carbon dioxide, and water are gathered for the first stage of photosynthesis which is called light reaction. The second stage of photosynthesis called the Calvin cycle produces glucose, oxygen, and other organic molecules. The products of photosynthesis are then used in the mitochondria to break down glucose, oxygen, and organic molecules which releases ATP, heat energy, carbon dioxide, and water. As ATP and heat energy is used to perform work, carbon dioxide and water is used to carry out photosynthesis resulting the cycle to repeat.

The two most important sequences on the plasmid that will be used are the ORI bacteria and the antibiotic resistance. The ORI is the origin of the bacteria and is an important part of the bacteria because it does the job of replication. The antibiotic resistance is equally important because it is used to see which bacteria take in the plasmid. In the lab there will be two micro test tubes used. One tube will be labeled +plasmid and the second tube will be labeled -plasmid. Both test tubes will contain the E. coli bacteria and 0.25mL of calcium chloride (CaCl2), but only the +plasmid tube will receive 10pL of pGLO plasmid. There will also be four agar dishes used in this experiment. They will be labeled as follows: LB/Amp+, LB/Amp-, LB+, and LB-. The LB/Amp+ dish will have luria broth, ampicillin, and the pGLO plasmid. The LB/Amp- dish will contain luria broth, ampicillin, and will not contain any plasmid. LB+ also contains luria broth, will not contain ampicillin and will contain the plasmid. The last dish, LB-, will have the luria broth, and will not have neither ampicillin nor the plasmid inserted. The LB/Amp- and LB- dishes act as the control group because they will be used to observe changes that occur without the plasmid. The LB/Amp+ and LB+ will act as the experimental dishes because the plasmid will

After the incubation, 1.5 mL of each of the three cultures were added to eppendorf tubes and centrifuged at 13,200 rpm for 1 minute. An alkaline lysis procedure like that of Birnboim and Doly was then performed to extract the plasmid DNA with 200 μl of alkaline SDS detergent solution (Birnboim & Doly, 1979). After

In experiment of Bacterial plasmid DNA extraction, solution 1 which consists of ice-cold resuspension buffer helped corrupting the cell membrane. Because cell membrane has not been broken down yet the collected blend is clear. However, later on inclusion of Solution 2 which is lysis buffer the cell membrane was broken. The solution 3 that is a neutralization buffer was added and it provided the contaminated genomic DNA to precipitate. Using centrifugation method provided the plasmid DNA to isolate from cell debris and genomic DNA. Protein contamination was tried to prevent using ethanol.

Once incubation is complete the viral DNA plasmid will be extracted from the cultures using a mini-prep protocol as follows. Cells will be washed twice with PBS and lysed in lysis solution (20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10 mM EDTA, and 0.2% SDS) for total genomic DNA extraction. The cell lysates will treated overnight with proteinase K (0.2 μg/ml), extracted with phenol-chloroform and precipitated with 2 volumes of 96% ethanol. DNA pellets will be dissolved in TE buffer containing 20 μg/ml RNase A and incubated for 1 hour at 37°C. The DNA will