Firstly, an amount of 40.90 g of NaCl was weighed using electronic balance (Adventurer™, Ohaus) and later was placed in a 500 ml beaker. Then, 6.05 g of Tris base, followed by 10.00 g of CTAB and 3.70 g of EDTA were added into the beaker. After that, 400 ml of sterilized distilled water, sdH2O was poured into the beaker to dissolve the substances. Then, the solution was stirred using the magnetic stirrer until the solution become crystal clear for about 3 hours on a hotplate stirrer (Lab Tech® LMS-1003). After the solution become clear, it was cool down to room temperature. Later, the solution was poured into 500 ml sterilized bottle. The bottle then was fully wrapped with aluminium foil to avoid from light. Next, 1 mL of 2-mercaptoethanol-β-mercapto was added into fully covered bottle. Lastly, the volume of the solution in the bottle was added with sdH2O until it reaches 500 ml. The bottle was labelled accordingly and was stored on chemical working bench. 3.2.2 Preparation of tissue samples for total genomic DNA extraction Firstly, samples were taken out carefully. The frozen tissues of sea cucumbers were thawed in the sink with running tap water followed by multiple washes using distilled water to remove the foreign particles. The surgical blades, surgical blade-holder, and labelled sample tubes were prepared. Then, the tissue samples were cut-sliced from each samples followed by storage in the properly labelled tubes. Each sample was stored in separate tube. Different blade was used for each sample. The obtained in-tube-samples were stored in -20 oC freezer for the next step of DNA extraction. 3.2.3 Total genomic DNA extraction using modified CTAB method (Doyle & Doyle, 1987) Total genomic DNA of Holothuria sp. samples were e... ... middle of paper ... ...erse) 5’-CCA GAG ATT AGA GGG AAT CAG TG-3’ (Palumbi et al., 1991). 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). The amplification process was started with pre-denaturation step using 96 oC for 7 min, followed by 30 cycles of denaturation at 95 oC for 1 min; annealing at 35 oC for 45 s and extension at 72 oC for 1 min 30 s, with a final extension at 72 oC for 10 min.
At this point the identity of the unknown compound was hypothesized to be calcium nitrate. In order to test this hypothesis, both the unknown compound and known compound were reacted with five different compounds and the results of those reactions were compared. It was important to compare the known and unknown compounds quantitatively as well to ensure that they were indeed the same compound. This was accomplished by reacting them both with a third compound which would produce an insoluble salt that could be filte...
We concluded that it is possible to examine small amounts of DNA by first replicating the sample using Polymerase Chain Reactions then using gel electrophoresis to. determine the genotype of the DNA. The main goal for our experiment was to learn how to examine DNA when there is only a small sample present. We examined the samples of DNA obtained from student hair using the replication method of Polymerase Chain Reaction and the inspection method of gel. electrophoresis.
These six samples (crude -/+, broken -/+, and whole -/+) were spun at 5000 rpm, and the resulting pellets were isolated and resuspended in DNase buffer. The set of suspensions labeled with a (+) was incubated in DNase enzyme for 15 minutes, and afterwards incubated in 15 uL of STOP solution. All six samples were lysed for DNA extraction with DNA extraction buffer, and micro-centrifuged at maximum speed. To precipitate the extracted DNA, the supernatants from each of the six samples were added to their correspondingly labeled micro-centrifuge tubes containing 7% ethanol (Parent et. al, 2008To bind the DNA, the ethanol lysate mixtures were transferred to labeled spin columns and spun for one minute in the micro-centrifuge at maximum speed. To wash the bound DNA, the spin columns were washed and spun three times at maximum speed. In order to elute the bound DNA, the samples were washed in 80 uL of distilled water and spun again for 2 minutes at maximum speed (Parent et. al,
Many things have impacted both the Science and Medical fields of study. Electrophoresis and DNA Sequencing are two of these things. Together they have simultaneously impacted both of these fields. On one hand, there is Electrophoresis. Electrophoresis is a specific method of separating molecules by their size through the application of an electric field. It causes molecules to migrate at a rate and distance dependent on their size. On the other hand, there is DNA Sequencing. DNA Sequencing is a technique used to determine the exact sequence of bases
Polymerase Chain reaction (PCR) is used to isolate a predetermined strand of DNA on the double helix. Once the desired DNA is isolated it is able to be copied as much as needed (2). In this experiment PCR was used to isoloate Vangl2 from Zebra fish embryos. In a PCR experiment, a primer is used to find and isolate the desired nucleotide sequence of DNA (2). In this experiment two primers were used as follows:
...spite benefits of the technique , that protocol cannot be used for other purposes such as PCR , because of presence of high amount of DMSO (7).DNA ladder assay is an easily available method and seems to be very useful for quick screening of apoptotic changes in cell populations. This method allows working with cell lysates and does not require any special laboratory equipment(10).The present study results showed that this DNA ladder assay used here, is a useful method for evaluating DNA damage and fragmentation caused by apoptotic agents , drugs , food additives and etc.
Briefly, cells obtained from a well isolated colony on an agar plate were resuspended in 50 l of cell lysis solution (.05 M Tris (pH 8), 1 mM 0.5M EDTA, 1% Triton X-100) contained in a 0.2ml PCR tube. The cell solution was lysed by incubation at 94℃ for 10 minutes in a T100 Thermal Cycler (Bio-Rad Laboratories, Irvine, CA). After centrifugation at 10000 × g for 5 minutes, 5 l of the supernatant was used as DNA template for amplification by the Polymerase Chain Reaction (PCR). Reaction mixtures consisted of DNA template, .25 µM each of 8F and 1492r primers (ref??), and 2X GoTaq Green Master Mix (Promega, Fitchburg, WI) in a final reaction volume of 25 µl. Amplification consisted of an initial denaturation step of 94oC for 5 min, followed by 30 cycles of 94oC for 1 min, 50oC for 1 min, and 72oC for 1 min 30 sec, with a final extension of 72oC for 5 min. Electrophoresis on 1% agarose gel under standard conditions was used to visualize an approximate 1600 base pair amplicon. DNA sequencing of the amplified 16S rRNA gene was performed by MCLabs (South San Francisco, CA) followed by analysis using the Basic Local Alignment Search Tool (BLAST) to make genus and species
Polymerase Chain Reaction (PCR) was performed to purify the DNA extract. A mastermix was needed to be made for the PCR products, the mastermix volumes were calculated and shown in table 1. PCR is a simple and inexpensive tool needed to focus on a segment of DNA and a copy it a billion times over. (2) This was needed to purify the DNA samples of the patients which were needed in a gel electrophoresis procedure. The agrose gel electrophoresis process uses electricity to separate DNA fragments by size as they migrate through a gel matrix. (3) Nucleic acid molecules are separated by applying electric field to move a negatively charged molecule through the agrose gel towards the positive charge. (3) The shorter the molecule the faster it travels compared to the larger molecules, because smaller molecules migrate more easily through the pores of the gel. (3) This accurately
Caplan M. Geralyn. DNA Isolation Lab. Owensboro Community & Technical College, n.d. Web. 5 April 2014.
The diversity and the unity of life are equally meaningful and striking aspects of our Earth (Dobzhansky, 1973). Although an astounding 1.2 million species have already been identified, it is estimated that another 8.7 million are yet to be discovered and classified (Mora et al., 2011). By understanding what unifies us –our genes, our understanding of the organisms we share our planet with will continue to grow.
A condenser and heat reflux was used to prevent reagents from escaping. Then the solid product was vacuum filtered. The product was recrystallized to purify it and the unknown
One of the most common technique used till date, is by using restriction enzymes to cut the DNA into fragments and then run on gel electrophoresis for separation according to their lengths. As we know that a single strand of plant DNA or animal DNA contain tens of thousands of genes, each working for the production of a specific protein essential for the growth and survival of the organism. PCR (polymerase chain reaction) can also be used for the amplification of genes segments which can be isolated through same procedure as gel electrophoresis. Selectable markers are used for the identification and The DNA Band at the correct size should be the one containing the gene, and it can then be excised from the gel. The need of an hour is to isolate individual gene and determine its function in shorter span.
TAE buffer (Tris-acetate-EDTA) 50X is use in this experiment of nucleic acids’ electrophoresis whether in agarose or polyacrylamide gels (Bisen, 2014). Usually for resolution of RNA and DNA fragment larger than 1500 bp TAE buffer is preferred, for genomic DNA and for large supercoiled DNA. In addition, TAE buffer contain low buffering capacity. Therefore, during prolonged electrophoresis, replacement (periodic) of the buffer is recommended. Furthermore, TBE buffer and also known as Tris-borate-EDTA 10X commonly used in polyacrylamide gel electrophoresis for the DNA and RNA (Kumar & Garg, 2005). TBE buffer is also used for agarose gels but it is rarely used for preparative gels for recovery of nucleic acids. TBE buffer will act as a strong inhibitor for nucleases. Double stranded linear nucleic a...
The sample was subjected to steam distillation as illustrated in Figure 1. A total of 50ml of distillate was collected while recording the temperature for every 5.0 ml of distillate. The distillate was transferred into a 250ml Erlenmeyer flask and 3.0 g of NaCl was added. The flask was cooled and the content was transferred into a 250-ml separatory funnel. Then 25.0ml of hexane was added and the mixture was shaken for 5 minutes with occasional venting. The aqueous layer was discarded and the organic layer was left inside. About 25.0ml of 10% NaOH was then added and the mixture was shaken as before. The aqueous layer was collected and then cooled in an ice bath. It was then acidified with enough 6.00 M HCl while the pH is being monitored with red litmus paper. Another 25.0 ml of hexane was added and the mixture was shaken as before. The hexane extract was saved and a small amount of anhydrous sodium sulfate was added. The mixture was then swirled for a couple of minutes then filtered. A small amount of the final extracted was tested separately with 1% FeCl3 and Bayer’s reagent.
In this experiment three different equations were used and they are the Stoichiometry of Titration Reaction, Converting mL to L, and Calculating the Molarity of NaOH and HCl (Lab Guide pg. 142 and 143).