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DNA Extraction From Chicken Liver

Deoxyribonucleic acid (DNA) is the hereditary material in humans and almost all other organisms. Nearly every cell in a person’s body has the same DNA. Most DNA is located in the cell nucleus (where it is called nuclear DNA), but a small amount of DNA can also be found in the mitochondria (where it is called mitochondrial DNA or mtDNA).
The information in DNA is stored as a code made up of four chemical bases: adenine (A), guanine (G), cytosine (C), and thymine (T). Human DNA consists of about 3 billion bases, and more than 99 percent of those bases are the same in all people. The order, or sequence, of these bases determines the information available for building and maintaining an organism, similar to the way in which letters of the alphabet appear in a certain order to form words and sentences.
DNA bases pair up with each other, A with T and C with G, to form units called base pairs. Each base is also attached to a sugar molecule and a phosphate molecule. Together, a base, sugar, and phosphate are called a nucleotide. Nucleotides are arranged in two long strands that form a spiral called a double helix. The structure of the double helix is somewhat like a ladder, with the base pairs forming the ladder’s rungs and the sugar and phosphate molecules forming the vertical sidepieces of the ladder.
An important property of DNA is that it can replicate, or make copies of itself. Each strand of DNA in the double helix can serve as a pattern for duplicating the sequence of bases. This is critical when cells divide because each new cell needs to have an exact copy of the DNA present in the old cell.
The extraction of DNA from cells and its purification are of primary importance to the field of biotechnology and forensics. Extraction and purification of DNA are the first steps in the analysis and manipulation of DNA that allow scientists to detect genetic disorders, produce DNA fingerprints of individuals, and even create genetically engineered organisms that can produce beneficial products such as insulin, antibiotics, and hormones.Â
Once the DNA has been isolated, it is essential to accurately determine its concentration for subsequent manipulation such as cloning or sequence determination.
To quantify the amount of DNA that extracted by using spectrophotometry.
The aims of this experience is to:
To use the properties of DNA to isolate long strands of DNA from liver cells.
To determine the yield of DNA isolated from a given amount of tissue.
To examine the light absorbing properties of purified DNA.
To examne the relationship between the concentration of a DNA solution and the absorbnce at 595nm of DNA-diphenylamine solution.
To generate a standrad curve relating DNA concentraton with the absorbance of DNA-diphenylamine solutions.
To use a standard curve to determine the concentration of an unknown DNA solution.
Materials and Methods As per lab manual.
Results Firstly, the chicken liver cell homogenate is treated with a salt solution such as NaCl and a detergent solution containing the compound SDS (sodiumdodecyl sulfate). These solutions break down and emulsify the fat

Fractional Distillation Process To Separate Organic Liquids

Distillation is a very useful method of purifiying liquids. Simple distillation is used when a pure solvent is required to be obtained from the solution. This is usually water. On the other hand, fractional distilation is basically used for the separation of a mixture of two miscible organic liquids having different boiling points. A common example of two liquids that mix with each other are ethanol and water. Another example is petrol and paraffin.
In this experiment, a mixture of acetone and toluene was provided. Basically, the liquid mixture was boiled to evaporate the liquid that had the lowest boiling point, referred to as the first fraction. The vapour passed up through a fractionating column, which is not used in a simple distillation. As the mixture vaopur passed up the fractionating column, it continually condensed and evaporated. This caused it to become increasingly richer in the liquid with the lowest boiling point until the vapour that reached the top consisted almost entirely of the component with the lowest boiling point. The vapour is then cooled in the condenser and so it condensed back to a liquid, which was collected, hence referred to as the distillate. When almost all the liquid with the lowest boiling point was distilled over, the temperature rised rapidly showing that a mixture of both liquids was distilling over. This should be collected in a separate container and discarded. Once the temperature reached the boiling point of the second liquid, the liquid was then distilled into another container.
This basically explained the process of fractional distillation. However, there is a theory behind all this, because the process of distillation should be related in reference to an ideal liquid mixture where one is more volatile than the other. Regarding the mixture of acetone/toluene provided in this experiment, ideal behaviour was assumed and once the process was carried out, the more volatile liquid was found by finding the boiling point of each component. It was noted that the more volatile liquid was acetone since this had a lower boiling point. This was discussed further in relation to boiling point-composition graphs.
2. Method 2.1 Chemicals used
Reagent:
Grade:
Manufacturer:
Acetone
GPR
BDH
Toluene
GPR
Merck
2,4-dinitrophenylhydrazine
GPR
Riedel de Haem
Sodium hydroxide
GPR
Tinstar
Iodine
GPR
BDH
Dioxane
GPR
Aldrich
A mixture of acteone (BDH, GPR) and toluene (Merck, GPR).
2.2 Apparatus
Fractionating column, thermometer, 100 mL round bottomed flask as the distillation pot, glass beads, anti-bumping granules, cotton wool, tight clip, Leibig condenser with rubber tubings, heating mantle, connecting side-arms as part of the fractional distillation setup, retort stand with clamp, water supply, 10 mL and 100 mL measuring cylinders, electronic balance, test-tubes, distilled water.
2.3 Procedure
Part a) The separation of the acetone/toluene mixture and the measurement of the boiling points of each.
The apparatus for fractional distillation was set-up appropriately using a 100 mL round-bottomed flask, the fractionating column provided, insulated well with cotton wool.
50 mL of the acetone/toluene mixture was placed in the 100 mL round-bottomed flask. This was measured using a measuring cylinder.
A few boiling chips or anti-bumping granules, which were small irregularly pieces of material, were added to the round-bottomed flask in order to allow prolonged boiling.
The apparatus was clamped accordingly from the neck of the round-bottomed flask and checked to be balanced and well set-up before the heating mantle was switched on.
Then round-bottomed flask was heated slowly using a heating mantle, until the reading on the thermometer reached a steady state and drops were observed to condense out of the Leibig condenser. This was the boiling point of the first fraction. This steady state temperature was recorded and the distillate was collected in a 100 mL measuring cylinder.
The distillation was allowed to proceed until no more liquid got out of the condenser into the measuring cylinder. Then the volume of the first fraction was recorded.
When all of the first fraction was distilled out, the temperature at the top of the column was observed to increase and then reached a second steady state, which was the boiling point of the second fraction. Drops of the second fraction were observed to start to condense out of the Leibig condenser. This steady state temperature which was the boiling point of the second fraction, was recorded.
The second distillate was collected in a clean measuring cylinder and then its volume was recorded.
Part b) The usage of two different tests for the identification of the liquid distillate having a carbonyl group. (acetone)
2,4-DNPH test was first carried out. 2-3 drops of the liquid to be tested were added to 3 mL of 2,4-dinitrophenylhydrazine, and shaken. Any observations and inferences were recorded.
The iodofrom test was then carried out. 4 micro drops of the liquid to be tested were dissolved in 2 mL distilled water, in a test-tube. The drops were added carefully using a pipette. 2 mL of 10 % sodium hydroxide were then added together with 2 mL of iodine solution, which were added slowly by drops. The substance was insoluble in water and therefore 2 mL dioxane were added. This was done so that the substance dissolved. Any observations and inferences were then recorded.
Each test was carried out twice, for each distillate.
Diagram:
The set-up apparatus for Fractional Distillation.
Precautions
It was ensured that the thermometre was positioned accordingly at the mouth opening of the Leibig condenser, where it indicated which fraction was being evaporated by noting the temperature readings. The thermometer position was very important because if the thermometer bulb was to be placed too high, the vapours would reach it before they pass into the side-arm to be collected, and the observed boiling point would be lower than it should be. If the thermometer bulb was to be placed too low, vapours of impurities might reach it, and a high reading for the boiling point range would be given.
It was ensured that the fractionating column was filled and packed with glass beads, for maximum possible surface area for vapour to condense on.
It was ensured that a joint clip was used and attached between the end of the condenser and the side arm so that there was complete attachment of the setup together and any spillage of the liquid distillate was prevented, but allowed to drop only from the side arm tube, where the vent was present.
It was ensured that the Leibig condenser provided was set-up accordingly with opening below meant for water to be pumped in while the opening at the top meant for water to be pumped out, and vice-versa. Although water pressure transfers from a higher to a lower height, if the condenser had to be the other way round the liquid might not be cooled completely as it would only condense the top portion of the condenser. Therefore if the rest of the part of the Leibig condenser was not cooled, the liquid would might evaporate into gas again at the bottom part of the condenser. This explained the importance of correct set-up.
It was ensured that as much of the second fraction as possible was collected, however at the same time care was taken so as not to allow the distillation pot, i.e. the 100 mL round-bottomed flask, to boil dry otherwise the residues might ignite or expode.
It was ensured that anti-bumping granules were used. These were placed in the 100 mL round-bottomed flask with the 50 mL of the acetone/toluene mixture. The granules were important since they allowed prolonged, smoother boiling without bumping and continuous even formation and release of vapour bubbles were observed.
It was ensured that cotton wool was used so that the whole fractionating column was completely wrapped and covered for insulation or lagging. This was important so that the apparatus remained as warm as possible and excessive cooling was avoided, but occurred very slowly.
It was ensured that parallax errors were avoided as much as possible by looking normally to the scale of the measuring cylinder were when taking readings of the volumes of liquids, or when taking temperature readings from the thermometre.
For safety measures, it was ensured that care was taken when distilling organic solvents in order to avoid explosions and fires. Hence, it was ensured that the vapour did not come into contact with flames, sources of sparks or very hot surfaces such as hot plates.
It was ensured that the apparatus was not completely sealed. A vent in the system was required so as to prevent pressure build up in the system as heating was carried out. Otherwise the apparatus would simply blow apart. Therefore, for safey measures, it was ensured that heating in a closed system was avoided.
3. Results Volume of acetone and toluene mixture used was: 50.0 mL

Observations

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