Essay On Whole Genome Sequencing

The following results helped obtain the haplogroup that in which the sequence of mtDNA would identify. The PCR reaction worked, and this can be determined by looking at the agarose gel in figure 1. If the PCR reaction was successful, than a band should appear around 550bp. Individual AC displays a band around 550bp, this means the PCR reaction was successful. The band for individual AC, depicts a low concentration of product, because the band faint. After the purification process the concentration, A260/280 ratio, and A260/A230 ratio were determined by using the nanodrop. The concentration of mtDNA in the product was 60.9 ng/uL. The ratio for A260/280 was 1.79 and the ratio for A260/230 was 0.77. The A260 and 280 are a spectrometer measurement that measure absorbance at wavelengths of

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.

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

Four microcentrifuge tubes were placed in a rack, labeled and numbered, in order to identify the group and the DNA/restriction enzyme that it held. Each of the tubes initially received 10 microliters of reaction buffer. There were two samples of suspect DNA provided along with two restriction enzymes (EcoRI and HindIII). Tubes labeled 1 and 2 received 15 μL of DNA from suspect one while tubes 3 and 4 received 15 μL of DNA from suspect two. Following that, 15 μL of Enzyme 1 (EcoRI) were added to tubes 1 and 3, and 15 μL of Enzyme 2 (HindIII) were added to tubes 2 and 4. (Table 1). The tubes were then gently tapped on the counter to mix the DNA and enzyme solution followed by incubation at 37°C for 45 minutes. After incubation, 5 μL of 10x gel loading dye were added to each of the four tubes of suspect DNA. The tubes were then placed on ice while the gel was under preparation.

To do this, 50 ml of buffer solution was added to 0.4 g of agarose in order to create a 0.8% agarose gel in a flask. The flask was placed inside microwave for intervals of first, 20 seconds then swirled, next 30 seconds then a minute and swirled. Then, while the solution was still hot, ethidium bromide (EtBr) was added in order to stain the gel. By staining the gel with EtBr, it allows the DNA to be visible under UV light once the process of electrophoresis has occurred. This is because the staining agent attaches only to DNA fragments.

Analysis of DNA from practicals 1 and 2 using the technique of agarose gel electrophoresis and analysis of transfomed E. coli from practical 2 (part B)

The different machinery that we used included a thermal cycler, agarose gel electrophoresis and a transilluminator. A thermal cycler is the instrument that is used that gives us the exact temperatures needed to complete the PCR. The thermal cycler is able to be used in cloning, sequencing, analysis as well as genotyping (3). The agarose gel electrophoresis is a way

Agarose gel was prepared to use for the detection of geneomic DNA by adding 1gm of agarose to 100 ml of 1X TBE buffer and it is dissolved by heating at boiling temperature. Then the agarose was left to cool at 55C°, before pouring in a casting plate to solidify. A required comb was placed near one edge of the gel, and the gel was left to cast. 1XTBE was poured into the gel tank and the gel plate was placed horizontally in an electrophoresis tank. The DNA samples were prepared by adding 1µl of loading buffer and mixed with 5µl DNA samples, and then the samples were added carefully to individual wells. Power was turned on at 45V for 15minute and 85V for 1 hours to run DNA or at 5-8v/cm. Agarose gels were stained with ethidium bromide by immersing

The gel was covered with a buffer and then six samples labeled A-F were deposited into the wells using a micropipette. Three of these samples (A, B, C) were control samples to compare to D, E, and F (the mother, child, and father). The safety cover was placed on the unit and then brought to a power source. The leads were connected the chamber and left for approximately 20 minutes. The agarose gel was removed from the tray and placed onto a sheet of plastic wrap. An Ethidium Bromide card was placed face down onto the gel to stain for approximately 5 minutes. Finally, the card was removed and the gel was placed on top of a UV light. The samples were pushed towards the center due to opposite electric charges. Agarose gel separates the DNA samples by the way they were cut. The restriction enzyme MST II cuts the DNA strand at CC/TNAGG where N is any nucleotide base. If the enzyme recognizes this, it is cut. If it does not recognize it then the strand is left whole. We were able to observe the DNA strands due to them being dyed and placed over a UV light. The control samples were utilized so that the other samples could be compared to test for their genotype. The data was analyzed in this way to differentiate between the different genotypes and the number of bars they

The initial step of Sanger sequencing, designing primer pairs for PCR, is often performed manually, with the aid of software such as Primer-Blast that can analyse only one genetic locus at a time, or PCR Suite, that can analyse more than one locus, but does not check for specificity. The manual design of primer pairs is especially cumbersome and prone to errors for long lists of genetic loci.

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.

Total RNA was extracted using the Trizol extraction kit (Invitrogen, Carlsbad, CA). First-Strand Synthesis System for RT-PCR (Invitrogen) was used to synthesize cDNA from 1.5 μg total RNA according to the oligo (dT) version of the protocol. Real-time PCR was performed using CFX Fast real-time PCR system (Bio-Rad Laboratories, Inc., Hercules, CA). The following cycle parameters were used for all experiments: 20s at 94°C, 30s at 60°C, and 30s at 72°C for a total of 45 cycles. The relative level of mRNA for a specific gene was normalized to GAPDH levels. Table 1 shows the sequences for all primer sets used in these

The chemical and reagents used for the extraction and quantitation of DNA were: Plant DNAzol (0.3ml/0.1g), 100% ethanol (100%: 0.225 ml/0.1 g, 75%: 0.3 ml/0.1 g), Chloroform (0.3 ml/0.1 g), Plant DNAzol-ethanol solution: Plant DNAzol, 100% ethanol (1:0.75 v/v), TE buffer (10 mM Tris, 1 mM EDTA pH 8.0), 1.2% agarose gel (Agarose, 1X TAE buffer), 6X loading buffer (glycerol, Tris/EDTA pH 8.0, ethidium bromide), .25X TAE buffer, Restriction enzymes and Restriction endonuclease buffers. All the chemicals used were quality grade. The restriction

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

Reference primers (PowerUp SYBR Green Fast PCR master mix, forward primer, reverse primer and ultrapure water).