Ccr And Dna Isolation Lab Report

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

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.

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

Our materials for the extraction of DNA included: the screwcap tubes, capless tubes, pipet tips, disposable transfer pipets, a foam microtube holder, a DI water bottle, a sharpie to label, a hot water bath, Non-GMO food, Test food, and a centrifuge. Our materials for the setup of the PCR reactions included: PCR tubes, screwcap tubes, pipet tips, an aerosol barrier, a foam microtube holder, a PCR tube holder, a 2-20 microliter micropipette, an ice bath, a sharpie to label, a plastic bag, a styrofoam cup with ice water, and a black tray with ice water. Finally, the materials for the electrophoresis of the PCR products included: Agarose gel, PCR sample tubes from previous labs, PCR tube holders, running buffers (300-350 ml), loading dye, a PCR molecular weight

Part three, first we made 2% agarose gel and let it harden while preparing our samples to load. Second, we took 10µL of our PCR reaction and put it into a tube labeled undigested and repeated this for a digested tube. Third, we added 1µL of HAEIII enzyme to the digested tube and placed in 37º water bath for 30 minutes so that it could digest. Once all done we loaded our samples to run through the agarose gel.

Unfortunately the agarose gel we ran for our PCR product as well as the plasmid vector was damaged when being carried to the light box in the back room and was not

I added 1 g of the agarose powder to 100 ml of TAE buffer in an Erlenmeyer flask. Using a magnetic stir bar, the agarose is heated until it is a completely clear solution. The agarose is poured into the chamber to make wells deep enough to hold 30 µl of solution. I inserted the comb in the chamber and waited for the gel to harden. To prepare for the samples, 10 µl of the XC loading buffer was added into the PCR tube. The professor demonstrated the techniques to load the gel with the DNA samples. After learning the techniques, I added my PCR sample in one the the wells. To run the gel, the power supply was set to 100V for approximately 30 minutes. To stain the gel, the gel was removed from the electrophoresis chamber and was placed into the FastBlast staining tray. The gel was placed in the staining tray for 2 minutes. Then the gel was rinsed for 10 seconds in Wash Tray 1. After rinsing for 10 seconds, the gel was washed for 5 minutes in Wash Tray 2. After agitating the gel for every minute in Wash Tray 2, the gel was washed for another 5 minutes in Wash Tray

The 2% agarose was prepared by dissolving 2g of agarose (Sigma NA grade) in 98ml of sterile H₂0, the solution was then heated until dissolved and allowed to cool to 55°C before adding 2ml of 50X tris-acetate-EDTA (TAE) buffer and 10µl of gel red™ Nucleic acid stain. The gel was then poured into a gel tray with a 20µl slot comb already fitted. All visible air bubbles were removed and the gel allowed to set.

TBE buffer ➢ gel loading dye (Type III) ➢ molecular biology grade agarose ➢ DNA sample or PCR samples Procedure: A. Making the gel (0.8% gel, 50 ml

800μL of sterile water and 200μL of the DNA isolate were pipetted into a 1mL UV-transparent curvette and mixed well. 1mL of sterile water was added to a second 1mL curvette. On the spectrophotometer, a baseline correction was done and the absorbance spectrum of the DNA isolate was recorded. A PCR master mix was made. 5μL of DNA isolate was pipetted into two new labelled PCR tubes. 5μL of Milli-Q water were pipetted into a third labelled PCR tube. 20μL of the created master mix was pipetted into one PCR tube and 20μL of the given master mix was pipetted into the other PCR tube. PCR tubes were placed in a thermocycler, and then frozen. Tube identification was noted. Electrophoresis Gel apparatus was set up using 1% molten agarose. 3μL of each of the three PCR tubes were pipetted into 3 labelled microcentrifuge tubes. 6μ of sterile water and 1μL of 10x loading dye was added to each. Gel was loaded with samples and a DNA ladder and gel was run. An image was captured and interpreted. Sample with the largest quantity of DNA, was prepared for sequencing. 10μL of

In the PCR based technique the time required to analyze a sample was significantly reduced (Weber and May 388). The major advantage of PCR is that it imitates the process of DNA replication, but limits it to specific DNA sequences of interest. In order for this process to occur, two primers, an enzyme DNA polymerase, excess of the four deoxyribonucleotide triphosphates, and a template DNA should be present. Several cycles of three major steps have to take place to produce the amplification. These steps are: DNA denaturation, primer annealing, and primer

Gene probes glow under UV light and have a fluorescent chemical attached to a short section of single stranded DNA. The chemical is then added to a DNA strand which contains a complimentary base sequence to a gene responsible for coding for a particular disease, such as cystic fibrosis. If the gene that is being looked for is present, then the gene probe will fix to it as it has the same complimentary base sequence. Once the gel is under UV light it will glow where the gene is present showing that the specific sequence is in the DNA tested. (5) Gel electrophoresis can be used to create a DNA profile for forensic purposes, paternity testing, evolutionary associations amid organisms and to look for genes associated with a

The results from this experiment are inconclusive as the 1% agarose gel using 142S primers did not display any bands (data not shown). The 142S transgene should be present in the 142S line only. In the control gel (Fig 3), there are bands in every lane; actin is used as the loading control, showing the PCR and gels were run according to standard procedures.

The PCR product did not appear in the no-template negative control reaction. This was fortunate, as it’s easy to contaminate the reaction with foreign DNA during purification and amplification. The control functions to show that

(A) Agarose gel analysis of DNA products from RT-PCR of CHIKV samples 1.100 bp DNA ladder, 2.CHIKV positive control, 3–5 patient sample, 6. Negative control.