Essay On Soil Detection In Soil

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

This was completed with over 65 tissue samples from various locations. The analysis was performed with quantitative PCR, using fluorescent signals.

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).

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 .

There are three steps in PCR, and they are; (1) Denaturing- DNA molecules are heated and separated into two single strands. (2) Annealing- A primer is used to start the process of building a new strand of DNA. (3) Extension- dNTPs are added to the reaction mixture to build a new complementary strand to the template strand. By using a primer a new strand grows in 5’ to 3’ direction and the template is 3’ to 5’ and they can be amplified by the original template.

PCR is an efficient molecular genetic diagnostic process. However, there are 4 main reasons that can make this diagnosis failing to give a precise result, which are the carry of PCR inhibitors in sample, the lack of optimization of the PCR primer and probe design, the lack of investigation in standard curve and the inaccurate sample and reagent pipetting (reference).

The American consumer market is twenty nine per cent of world market aside from the basic essentials we need to sustain life. We need to breathe, eat and drink water but we consume a large amount of other resources and services that eventually contaminate the soil we live on. There are so many new inventions and with everyone trying to have the next best thing that allows us to pollute more and waste more into the soil. In this paper I will focus on: What is soil contamination, the type of toxins found in soil contaminated areas and solutions on how to lower the chances of soil contamination.

Being able to determine the amount of DNA present in an organism has been successfully done using qPCR. In this experiment, qPCR was used to identify how much fungi could infect mutant and wildtype lines of A. thaliana. Also, qPCR was used to amplify the 16s rRNA ITS (internal transcribed spacer) of an unknown fungal pathogen. Other ways qPCR has been used in recent years include the use of qPCR to quantify copy number variants in the HER-2 gene which is a proto-oncogene. This method was particularly employed in the formalin-fixed-parafilm-embedded tissues - due to the fact that their DNA is usually broken into smaller pieces – to gain accurate results. This study concluded that qPCR produced similar results with already existing

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

This essay based on the principle of real time PCR which uses CYBR green dye that combines to any double stand DNA. This process included two maim steps. The first step was designing of HSV-1 primer and /or HSV-2 primer (we have chosen only HSV-1). BioEdit software has been used to edit the nuclide sequences where necessary. After that the primer has been ordered. The second step was in the laboratory which included applying the HSV-1 primers to real time PCR protocol. The final results of this protocols aim to demonstrate the quality of HSV-1 primer that we have designed by interpretation three criteria. These are specificity, efficiency and

Extracted RNA (1 μg) was reverse transcribed using high-capacity cDNA reverse transcription kit, supplied by Applied Biosystems, USA according to the manufacturer's instructions. The cDNA product was diluted in 200 μL Nuclease-free H2O.

• Make single-strand cDNA from mRNA – downstream antisense primer or random hexamer and RT to make complete cDNA copy of RNA molecule • Use cDNA, DNA polymerase, and a downstream primer in conventional PCR – extension leads to double-stranded DNA • Regular PCR of dsDNA

Further, to measure the transcript levels of foxh1 in X. tropicalis tissue fragments (early gastrulae stage) and transcription levels of the members of PouV family, the quantitative RT-PCR was used. While this method is known as a sensitive and powerful tool for analyzing of RNA samples and it has a remarkable potential for quantitative applications, its technical features require a complete knowledge of the system. Experimental variations in individual RT and PCR efficiencies should be corrected in the quantitative RT-PCR. Moreover, the expression of one or multiple housekeeping genes is essential as a reference for the analysis of testing genes’ expression levels. One of the other strong points of this research is, in fact, having appropriate control genes in the study. For example in the associated results for Figure 1-A, the transcription level of the ribosomal protein L1 (rpl1) i.e. the control gene is clearly provided.

M. Sc. in Microbiology, Division of Microbiology, Faculty of Agriculture, Annamalai University, Annamalai Nagar, Tamil Nadu