T-RFLP and AMF community structure analysis
T-RFLP was performed in twelve samples by targeting larger subunit (LSU) of AMF rDNA with specific primers. Nested PCR was performed with two set of primers, LR1/FLR2 was used for the first round PCR. The resulting PCR product was diluted 20 folds (using TE buffer) and used as a template for second round PCR with FLR3/FLR4 (Gollotte et al. 2004). Courtney et al. (2012) proved that nested PCR amplification of AMF large subunit with FLR3/FLR4 followed by AluI and MboI digestion was found to be effective in detecting AMF species present very low frequency in soil. FLR3/FLR4 was fluorescently labeled at 5’ end with FAM and HEX, respectively. PCR reaction was comprised of 50 µl reaction mixture containing 1 µl of template DNA, 0.2 mM of each dNTP, 0.4 µM of each primer, 5 µl of 10x Taq buffer and 1.25 units of Taq DNA polymerase. Thermal cycles included one step initial denaturation for 5 min at 95oC, 25 cycles (30 cycles for nested PCR) consisting of 1 min at 95oC, 1 min at 56oC (58oC for nested PCR) and 1 min at 65oC followed by final extension for 10 min at 65oC. PCR products were purified using QIAquick PCR purification kit (QIAgen, Germany) with an elution product of 60 µl. Purified PCR products were separately digested with the restriction enzyme AluI and MboI (Promega, USA). Sixteen microliters of PCR product and five units of restriction enzyme in the manufacture’s recommended buffer were incubated for 4 h at 37oC for digestion. Terminal restriction fragments (T-RFs) of all samples were determined using ABI 3130 DNA sequencer with ROX 500 (Applied Biosystem, USA) as a standard. Genemapper ver 3.7 (Applied Biosystem, USA) was used to identify and quantify the 5’ labeled T-RFs.
T-RFs p...
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...(2.0μl) for PCR reaction (NS31-GC/Glo1 primers) with the same conditions as described above in order to test the existence of double bands. Eluted single bands were sequenced using NS31/Glo1 primers in automated ABI 3100 sequencer (Solgent, South Korea). Nucleotide sequence data obtained in this study were compared with those from the genbank (http://www.ncbi.nlm.nih.gov/genbank/) using the BlastN program (Altschul et al. 1997). The sequences were submitted in genbank under the accession number of KC887744- KC887757.
Statistical analysis
Pearson correlation coefficient analysis was performed using SPSS software ver. 19 to find out the relationship between HMM and AMF diversity. Data on spore count and molecular studies were subjected to analysis of variance (ANOVA). Significance at the level of 5.0% was tested by t test using SAS package, version 9.1.3 (SAS 2010).
The miniprep consisted of isolating the DNA plasmid from the bacterial cells. This was used to identify the success of EGFP ligation into pET41a(+) vector upon restriction digest and gel electrophoresis. Additionally, Polymerase Chain Reaction (PCR) was run on the isolated DNA plasmids with one of the primers specifically annealing to a part of pET41a(+) sequence and the other annealing to the EGFP gene.
The purpose of this study is to identify an unknown bacterium from a mixed culture, by conducting different biochemical tests. Bacteria are an integral part of our ecosystem. They can be found anywhere and identifying them becomes crucial to understanding their characteristics and their effects on other living things, especially humans. Biochemical testing helps us identify the microorganism present with great accuracy. The tests used in this experiment are rudimentary but are fundamental starting points for tests used in medical labs and helps students attain a better understanding of how tests are conducted in a real lab setting. The first step in this process is to use gram-staining technique to narrow down the unknown bacteria into one of the two big domains; gram-negative and gram-positive. Once the gram type is identified, biochemical tests are conducted to narrow down the specific bacterial species. These biochemical tests are process of elimination that relies on the bacteria’s ability to breakdown certain kinds of food sources, their respiratory abilities and other biochemical conditions found in nature.
The eighteenth exercise of the laboratory manual titled Unknown Identification and Bergey’s Manual is an experiment to identify an unknown bacterium. In this exercise, a student must randomly choose a numbered bacterium available to the class. The keys in Appendix H, located on the last pages of the book, are the major helpful tools in this exercise because it provides completed steps of tests that needs to be performed in order to distinguish certain bacteria. This means that in this exercise, various types of tests and techniques must be performed to identify the chosen unknown bacterium. The unknown bacterium that I selected was number thirty-nine in which I discovered as the Bacillus megaterium after conducting several tests.
When the PCR technique is completed, the tubes are stored at 4°C until analysis of the tubes. To analyze the PCR results with the gel electrophorese, 2.5ul of the 10x loading dye is added to each PCR reaction tube. The gel for the electrophorese consists of 1.5% agarose gel with 0.5x TBE and 200ng/ml ethidium. bromide. The sand is a sand.
Amplification reaction was done in a 25.0 µL reaction mixture containing 0.4 µL DNA (from DNA extraction), 5.0 µL of 10X PCR reaction buffer, 14.2 µL of sterelized dH2O, 2.0 µL of magnesium chloride (MgCl2, 25 mM), 1.0 µL nucleotide/dNTP mix (10 Mm), and 0.4 µL of 5 u/µL Taq DNA polymerase for each primer namely respectively. The components and the volume used for the amplification reactions are listed in Table 3.2. For the reaction, PCR reaction was performed in a programmable gradient-enabled thermocycler (Bio-Rad MyCycler™ Thermal Cycler).
It all began in and around the year 1919. Sula Peace, the daughter of Rekus who died when she was 3years old and Hannah, was a young and lonely girl of wild dreams. Sula was born in the same year as Nel, 1910. Sula was a heavy brown color and had large eyes with a birthmark that resembled a stemmed rose to some and many varied things to others. Nel Wright, the daughter of Helene and Wiley, was and unimaginative girl living in a very strict and manipulated life. Nel was lighter in color than Sula and could have passed for white if she had been a few shades lighter she. A trip to visit her dying great-grandmother in the south had a profound effect on Nel’s life. In many ways the trip made her realize her selfness and look at things around her in a different light, eventually sowing the seeds that initiated the friendship between herself and Sula. The two girls met each other at Garfield Primary School after knowing each other at a distance for over five years. Nel’s mother had told her that she could not interact with Sula because of Sula’s mother sooty ways. The intense and sudden friendship between them which was to last many years was originally cultivated my Nel. The period in history and the mentality of the people in their immediate surroundings played an impressive part in the formulation of the friendship between Sula and Nel. When they first met at school, it was as if they were always destined to be friends.
For the original analysis, the corrected pairwise distance will be calculated using the Jukes–Cantor and the Maximum Composite Likelihood Model. The Jukes–Cantor model assumes that the rate of nucleotide substitution or all nucleotides (C, A, T and G) are equal, that nucleotide frequencies are equal, that there is an equal rate of substitution among sites, and does not correct for the lower rate of transversion substitutes in comparison to transitional substitutions (Jukes and Cantor, 1969). The Maximum Composite Likelihood takes into account the phylogenic relationship between sequences, using the sum of the log likelihoods of the bases as the composite likelihood. Both pair wise distances and substitution parameters are estimated using the Maximum Composite Likelihood (Tamura et al. 2004). Both models should yield different maximum sequence divergence and average divergence that can then be compared to the original paper. With sequence divergence data, the temporal origin of the genus can be identified. The two alternate models to the Kimura-2 parameter will be analyzed to discuss which methods yield results closest to the expected time origin of the genus
...the total number of asci X 100. In order to calculate the map distance, it was necessary to divide the percentage of crossover asci by 2. This has to be performed because only half of the spores in each ascus are result of crossing over. Each student had to count, at least 100 spores, in order to determine if crossing over occurred in a particular perithecium. A data was collected to determine whether various environmental conditions affected the crossing over in Sordaria sp.
"Polymerase Chain Reaction (PCR) Fact Sheet." National Human Genome Research Institute. 10 Dec. 2007. National Institutes of Health. .
“David M. Lodge // Department of Biological Sciences // University of Notre Dame.” University of Notre Dame. 2009. Web.1 Mar. 2010.
In their life, at one point or another, people deny to themselves and others what they really feel and what really happened. Some people go on living their entire lives denying their true emotions. In Toni Morrison’s novel Sula, characters constantly denied their feelings and their actions. Sula Peace, her best friend Nel Wright, and Nel’s mother do not listen to their feelings and hide from their true emotions.
2)Campbell, Neil A., and Jane B. Reece. Biology. San Francisco, CA: Benjamin Cummings, 2008. Print.
Leboffe, M. J., & Pierce, B. E. (2010). Microbiology: Laboratory Theory and Application, Third Edition 3rd Edition (3rd Ed.). Morton Publishing
J. Losos, K. Mason, S. Singer, based on the work of P. Raven, & G. Johnson, Biology, 8th ed., (McGraw-Hill Education (Asia), Singapore, 2008), pp. 994-995.