Pcr Lab Report

Ps: the iodine was already really dark so it was very hard to see much difference between the control and the others.

Observation: no bugs were found except small, black, gnats were all close to the ground.

20 ul of DNA was added to 20ul of Master Mix. The Master Mix contained primers, dNTPs, Mg2+, Taq DNA polymerase, and yellow dye. Both the DNA and Master Mix were mixed with the micropipette. The DNA was then put into the thermal cycler containing 40 cycles of PCR amplification, amounting to 3.5 hours of amplification.

In this experiment, the E. coli sample used harbors a genetic mutation on the repressor gene of the lac operon, resulting in the constitutive enzyme production of -galactosidase. Therefore, the expectation of this strain of E. coli should produce higher yields of -galactosidase, as so much as 1000 fold in comparison to wildtype, non-mutated strains (Mowery and Seidman, pg. 3).

The use of HindIII restriction enzyme was expected to yield two pB325 DNA fragments, one 1914 base pairs, while the other at 5248 base pairs. To determine the lengths, a plasmid map was used. The results showed that lanes 2 and 4 (the traditional and kit restriction digests) had two pieces of DNA at ~2000 and ~5000 base pairs, while lanes 3 and 5 had 5 bands (Figure 1). These results agree with the hypothetical data and thus evidence shows that the experiment was successful. The isolated and confirmed pB325 plasmid DNA was later used for the transformation of BL21 E. coli

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

This experiment overall was very successful as using information obtained from the results of both the single and double digests, a credible restriction map for the unknown plasmid could be constructed. Within this experiment, both single digest and double digests consisting of three restriction endonucleases were used in order to map out the restriction sites of the enzymes making up an unknown plasmid. In order to separate the DNA fragments by their distinct number of base pairs, it was necessary to run an agarose gel electrophoresis. For this particular experiment, a 1% agarose gel was used as this concentration ultimately results in pores that can separate the DNA by size. The process of gel electrophoresis is made possible by the electric current that is used to move the samples of DNA throughout the gel. For this particular experiment, the gel was run at a current of 100 volts. As a result of the phosphate backbone of DNA, DNA is negatively charged. Because DNA is negatively charged, it moves away from the negative electrode and moves down the gel toward the positive electrode as it is attracted that way. What allows for the resistance the fragments face when moving away from the negative electrode is the texture of the gel itself. As a result, the slower fragments of DNA have the ability to move at a faster rate than the larger fragments of DNA. Each sample loaded into the gel contains a loading-dye for two reasons. One reason is that the loading dye contains

The infill brick walls of Specimen 5 were produced by the small windows opening. These window openings were shifted to the side of the one column at stories. Until the end of the experiment, 9 hysteresis cycles were applied to Specimen 5 at both forward and backward. Specimen 5 reached 9.14 kN lateral force and +7.56 mm displacement at 4 hysteresis forward cycle and -19.31 kN lateral force and -21.51 mm displacement at 8 hysteresis backward cycle. When Specimen 5 reached to ultimate lateral load-carrying capacity, interstory drift value was 0.7% at forward and interstory drift value was 3% at backward. Load controlled program and base shear versus second story displacement hysteresis curve of Specimen 5 are shown in Fig. 14.

DNA is a negative molecule so it will move towards our anode (positive end) as the small fragments will move faster and the large ones slower. With a DNA ladder, we could compare the size of the gene we needed to the marker with a known size. TOPO cloning along with transformation were then carried out. With help from DNA topoisomerase, TOPO cloning uses our gene from the PCR reaction and places it within our vector (pET101/D-TOPO) via directional cloning. Directional cloning allows us to control the direction of our insertion. During this procedure, we had to be careful not to mix too thoroughly in order to keep the overhangs necessary intact. Also a master mix containing salt allowed for a higher efficiency due to the fact that the salt prevents the topoisomerase from rebinding thus potentially nicking the DNA post ligation. Heat shock was then used in order to ensure that our plasmids were placed within the bacterial cells. The cells containing this plasmid could then be placed on an LB plate with S.O.C. medium containing ampicillin. The S.O.C. medium contains nutrients that allowed our transformed cells to survive and thrive. Ampicillin kills certain bacterial cells that do not contain a resistance towards it. Our new transformed cells had this resistance so it ensured that the colonies formed during incubation did in fact have the gene we were looking

There are five stages to this experiment: pre-incubation, incubation, heat shock, recovery and growth, and selection. First, two micro test tubes were obtained and labeled “+” and “-”. These labels were representative of transformed (+) and untransformed (-) Escherichia coli (E. coli). 250 microliters of transformation solution (calcium chloride) was added to the test tubes and placed on ice. During the pre-incubation period a single colony was placed into the test tube labeled “+” and was completely dispersed in the solution. This was repeated for the tube labeled “-“ as well. Then the solution was returned to the ice. In the incubation period, the pGLO DNA solution was added to the tube labeled “+” but was not added to the tube labeled “-“. The tube was then returned to the ice and incubated for 10 minutes. While this was undergoing the process, four agar plated were labeled according to what they had been treated with. The plates were treated accordingly: plate 1 contained lysogenic broth (LB)/ ampicillin (amp)/ plasmids with a green fluorescent protein (pGLO); plate 2 contained LB/amp/arabinose sugar with pGLO; plate 3 contained LB/amp without pGLO; and plate 4 contained LB without pGLO. The next step was heat chock. Both test tubes were placed in a water bath that was set at 42°C for 50 seconds. After 50 seconds the test tubes were rapidly transferred back to the ice were they

Beta-Galactosidase is a bacterial protein that is coded for the Beta-Galactosidase open reading frame, which is part of the greater overall Lac-Z gene. Beta-Galactosidase cleaves the sugar lactose for metabolic processes. If the homogenic substrate known as X-gal is added to the agar plate, the x-gal is hydrolyzed to form 5- bromo-4-chloro-indoxyl, which spontaneously dimerizes to produce an insoluble blue pigment called 5, 5’-dibromo- 4, 4’-dichloro-indigo and that produces a blue color (Padmananbhan, 2011). This blue color is a visual phenotypic indicator showing whether the beta-galactosidase open reading frame is being transcribed or not. Restriction enzymes are able to recognize specific palindromic sequences in DNA base pairs which are

Methods: Due to experimental error in previous labs, we did not obtain the DNA of our original taxa. We then chose to use DNA sequences supplied by the TA from the 2016 and 2017 school year. We picked six fungal taxa obtained from differing parts of English Yew Trees, including its fruit, bark, and leaves. Using the DNA sequences given, we copied the sequence into the BLAST system found on blast.ncbi.gov, making sure to add “fungi” into the organism box and to search for somewhat similar sequences. Once the program obtained the results, we scrolled down to the first result that gave a genus and species name, with an “ITS” in the description. Using the information given in the results, we filled in the information on Table 2, including seq

My group and I had to see if there were any signs of glucose when lactase was incubated in water at 80 degree Celsius for 5 minutes. After the incubation, we took out lactase and tested it for glucose. It came out negative. We all deducted that having the water at 80 degrees Celsius subjected the enzyme to an extreme temperature making the lactase show no sign of glucose because it was too hot for the enzyme to activate.

Plasmid DNA with Restriction Digest: The purpose of restriction digest of plasmid DNA is to understand how each DNA plasmids was cut with the given restriction enzymes and perform gel electrophoresis to observe the samples. Nine restriction digests were created, containing three digests for each of the three plasmid DNAs identifying as recombinant, non-recombinant, and unknown. Out of the nine digests, six are actual digests and three are undigested controls. A master mix is created to add to each of the nine samples with its following stock ingredients: 10 ul of 2X Reaction Buffer, 1 ul of Nco1, X ul of sterile water (Single digest), 10 ul of 2X Reaction Buffer, 10 ul plasmid DNA, 1 ul Nco1, 1 ul of Not1, and X ul of sterile water (Double

In the lab, we were introduced to six different DNA samples, the Crime Scene and 5 suspects. Our Independent variable of the experiment was the restriction enzyme, and our dependent variable was the DNA sample that matched the Crime Scene DNA. With our variables stated. This splices up the DNA into segments that are ready to begin gel electrophoresis.