Analysis Of Primer Sets

There were several steps used to acquire the colony necessary for the PCR. First a student forearm was swabbed using a cotton swab, the cells were then placed in an agar plate. DNA was then extracted from the cultured bacteria by using a technique to lyse the cells and solubilize the DNA, then enzymes were used to remove contaminating proteins. The DNA extraction consisted of a lysis buffer that contained high concentrations of salt for denaturing. Binding with the use of ethanol and a washing step to purify the DNA. The final step for the DNA extraction was elution where the pure DNA was release. Proceeding the extraction of DNA the results of the 16s gene amplification were examined through gel electrophoresis it was analyzed by estimating the size of the PCR bands with marker bands. After measuring the success of the extraction, a technique called TA cloning was started. Cloning of PCR products was done by using partially purified amplified products with

Figure 1 Gel Electrophoresis for Replication Taster PTC. The gel is composed of an ethidium bromide stained 3% agarose gel demonstrating DNA fragments which were a depiction of PCR amplification. The agarose gel contains nine loading samples, including from left to right, the MW marker lane 1 precision mol mass standard, lane 2 TB undigested PTC (5µl of DNA, 5µl of master mix P, and 2.5µl of loading dye), lane 3 TB digested PTC (5µl of DNA, 5µl of master mix P, 2µl Fnu4HI, and 3µl of loading dye), lane 4 TB A(L)DH G (10µl DNA, 10µl master mix G, and 5µl loading dye), lane 5 TB A(L)DH A (10µl DNA, 10µl master mix A, and 5µl loading dye), lane 6 MG undigested PTC (5µl of DNA, 5µl of master mix P, and 2.5µl of loading dye), lane 7 MG digested PTC (5µl of DNA, 5µl of master mix P, 2µl Fnu4HI, and 3µl of loading dye), lane 8 MG A(L)DH G (10µl DNA, 10µl master mix G, and 5µl loading dye), lane 9 MG A(L)DH A (10µl DNA, 10µl master mix A, and 5µl loading dye).

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.

The Basic Local Alignment Search Tool (BLAST) was the final portion of this lab report. BLAST is provided courtesy of the National Center for Biotechnology Information (NCBI). After running the five chosen tests, students were given the two 16S rRNA sequences that were associated with their assigned culture. The BLAST results served to confirm or disprove the hypothesis of what the unknown may be. The BLAST program compared the given 16S rRNA sequence to a database of known sequences and searched for similarity. (10) This search tool is capable of comparing nucleotide or protein sequences to find statistical significance between the matches. A perfect match to the unknown sequence is indicated by a

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

Also (5μl) of DNA template that extracted from stool samles was added then 1.5 μl of each type of Primers(forward and reverse)added to the master mix and then blend well using Exispin vortex centrifuge ,then this tubes would transferred to the Thermocycler machine, which has been programmed by the previous program for amplified of ITS1 region.The PCR products were electrophoresed in agarose gel and visualized on UV trans illuminator and then photographed using photo documentation .

NaOH is then applied for cell lysis and the ‘unzipping’ of dsDNA to ssDNA. The ssDNA may then be used to isolate and replicate the PCR product through the use of PCR and site specific primers, using 2 specific primers to isolate both sides and ends of the mtDNA D.loop, multiple runs of PCR are taken to receive multiple copies of the PCR product. The following sequence primers are used to isolate the PCR

The Blue sample, which was hypothesized to be recombinant, should have produced similar if not identical bands to the known recombinant Green sample, which is not the case in the PCR gel. Instead, the Blue sample shows faint bands more closely resembling the Red sample, known to be non-recombinant. The bands did not show through for the PCR samples which indicates that the PCR preparation was not successful. This is further confirmed when analyzing the positive control which produced no results. The lack of bands in the negative control suggest that failure this portion of the experiment was not due to

Then 5 µL of each PCR sample was loaded onto a 2% agarose gel in the following order: Bioneer 25/100bp mixed DNA ladder, WT CCR5 plasmid, ∆32 CCR5 plasmid, cheek DNA, negative control. The gel was allowed to develop.

With the sample wells near the cathode of the electrophoresis chamber, a potential difference of 100 volts was applied across the gel. The chamber was covered with aluminum foil to protect the SYBR SafeTM dye from light. Once the Orange G reached about 80% across the gel, the electrophoresis was stopped and the gel was carefully removed. In order to visualize the results of the PCR and electrophoresis, an image of the gel was taken under ultraviolet light. The resulting bands of the control samples were verified. Using the bands of the control samples as a guide, the presence of the Bt gene was evaluated by comparing the bands produced by the test corn chip.

The computing component of sequences searching has increased the pace of the research as comparison of genetic

In order for DNA samples from suspect one and suspect two to be digested by two different restriction enzymes, four reaction tubes were required, labeled 1-4. In each reaction tube, with a micropipette, ten µL of reaction buffer was used. All the samples were prepared based on the given chart (shown below as Table 1) (Upadhyaya, 2017, p. 58). As far as all four having the same enzymes that was the end, so to not cross-contaminate, the micropipettes tips had to be changed each time (Upadhyaya, 2017). The reaction tubes one and three contained 15µL of enzyme 1 and enzyme 2 was added to reaction tubes two and four. Then, reaction tubes one and two were filled with 15µL of

The article describes using the method of gel electrophoresis to separate DNA fragments to identify and analyze certain sequences. The DNA fragments were broken down using a restriction enzyme from Haemophilus influlenzae, allowing the researchers to see that these restriction enzymes can be used to study DNA and its structure. The first step of the gel electrophoresis was to set the gel matrix. Agarose was used to separate DNA molecules, and acrylamide was used to separate proteins. The gel starts off as a liquid, which was poured into a molding tray. A comb was placed in the liquid matrix so that when the matrix solidified, wells formed to load samples in them. After the gel solidified, the mold was removed and placed

DNA isolation is a process of DNA purification through physical and chemical means. DNA was protected within the nuclear membrane of B. rapa, which was surrounded by a cell membrane and cell wall. Mechanical and chemical lysis of the cell was necessary to break open the cells and solubilize the membranes to isolate the DNA. Mechanical lysis, which consisted of grinding the leaves with a pestle, broke down the cell wall. Chemical lysis, through the use of lysis solution, broke down the lipid-base membranes. The lysate was placed inside the Zymo-spin IV spin filter to remove chunks of debris from the solubilized cell components. A binding buffer was added to the collection tube that held DNA,

PCR reaction was carried out in a 25 μl reaction mixture under standard condition, contained 1x buffer, 0.2 mM of four dNTPS, 1.5mM mgcl2, 1 pM of forward (sequence) and reverse (sequence) primers and 50ng of genomic DNA. Initial denaturation at 95˚C for 4 min was followed by denaturing at 94˚C for 30 seconds, annealing at 60˚C for 30 seconds and extension at 72˚C for 30 seconds for 32 cycles. The final extension was 8 min. The amplicons were run on an ABI 3130x (Applied Biosystems, Foster City, California,