Ion-paired reverse phase liquid chromatography for the detection, separation and quantification of nucleotides If you are working in the field of molecular biology, there is hardly a day that goes by without the use of nucleotides. But beyond the use of the four well known deoxynucleotides in PCR, there are several other uses of nucleotides. In the field of enzymology, nucleotides are used as substrates of various enzymes. For example, kinases and phosphatases use nucleotides as substrates while phosphotransferases transfer phosphate group from one nucleotide substrate to another. If you want to study the kinetics of such enzymes, you need to quantify either the substrate or the product. Detection of nucleotides can be done in several ways. …show more content…
The only addition is the use of an ion-pairing agent. It is a compound that has a hydrophobic group which sticks to the stationary phase and a charged group which hangs out of the stationary phase, making it charged. So now the hydrophobic column has become polar. Ion-pairing agents are either acidic like tetrabutylammonium hydrogen sulfate or basic like sodium dodecyl sulfate. Acidic ion-pairing agents are positively charged and will bind anionic molecules while basic ion-pairing agents are negatively charged and will bind cationic molecules. The choice of ion-pairing agent will depend upon your experiment but there are a lot of options available out …show more content…
The two most important factors that can affect your separation are the type of organic solvent used and its concentration in the mobile phase. Two most widely used organic solvents are methanol and acetonitrile. Methanol is more polar than acetonitrile and hence is less powerful in separating compounds with stronger affinity to the stationary phase. However, this is not the only factor considered while choosing your organic solvent. Sometimes methanol just works better than acetonitrile in separating out a specific set of nucleotides. It would be wiser to try out both organic solvents and see which one works best for you. Another important factor is pH. Nucleotide separation is usually carried out at pH 6-8. At very low pH, nucleotides are not negatively charged and hence will not bind to the stationary phase. At very high pH, the ion-pairing agent will be neutral and hence not bind to the nucleotides. The concentration of ion-pairing agent should also be optimal in order to allow a higher binding capacity. Using a higher than optimal concentration will increase the affinity of the nucleotides for the stationary phase and make elution a difficult
The Solid sequencing platform, produced by Technologies/Applied Biosystems (ABI), performs sequencing by ligation method. Similar like the Roche 454 library preparation, genomic double strand DNA were sheared into small pieces and ligated with two types of adatptors P1 and P2 on two ends. One end with P1 adaptor binds onto the surface of the magnetic bead and emulsion PCR takes place to amplify single nucleotide fragment. Then the oil was washed out and four fluorescent labeled di-bases probes were added into the beads mixture. By matching the 1st and 2nd position of the template by di-base probes, fluorescence was detected and the extra tail with fluorescent probe is cleaved out.
The intermolecular forces and molecular structure of the molecules being separated determine the ideal solvent to use in chromatographic separation making sure that they each have the same
The second step performed was the Gram Stain test. The Gram Stain was used to determine whether the unknown bacterium was gram-positive or gram-negative. First, the unknown sample was smeared onto a slide along with a drop of distilled water. Second, the unknown was air dried and was heat fixed.
The key enzyme in the process is DNA polymerase that forms the PCR product by linking individual nucleotide bases (adenine, guanine, cytosine and thymine). Short DNA fragments with sequence complimentary to target DNA are called primers; these specify the DNA sequence that has to be amplified. The PCR reaction mixture is made in a 96 well plate or test tube and placed in a thermal cycler. The thermal cycler is a machine used to amplify DNA by altering the temperature in repeated cycles using three precise pre programmed steps: Denaturation – By increasing the temperature in the thermal cycler the DNA is denatured creating separate single stranded DNA
20ul 2. Turn on PCR machine. 3. Input sample identifiers on qPCR running software, including NTC, positive control and quantities of standards. 4.
The Detector: The separated ions are then measured, and the results displayed on a chart. Mass spectrometry has both qualitative and quantitative uses. These include determining the structure of a compound, quantifying the amount of a compound in a sample and determining the isotopic composition of elements in a molecule. This technique basically studies the effect of ionizing energy on molecules.
Intro: Separation and purification of an unknown/complex compound can be done by using techniques such as liquid-liquid extraction, solid-liquid extraction, recrystallization, melting point, and thin layer chromatography. In this experiment, these techniques were used to separate excedrin’s components containing caffeine, excedrin, and acetaminophen, Each component has its own chemical properties and characteristics such as polarity, reactivity, and solubility. Knowing how to separate and purify compounds from each other is an important skill within in a lab setting. A few techniques for first initially separating compounds apart are liquid-liquid extraction and solid liquid extraction. Liquid-Liquid extraction involves using a seperatory funnel and release on varying solubities and different solids in immiscible solvents.
7. In this experiment, if the sucrose concentration were increased to 70 g/l would you expect sucrase activity to be significantly higher than the activity at 35 g/l. Explain your answer. No, because based on the results once it reached 30 g/l 35 g/l the results had stayed the same. There, the activity is lessening and coming to what looks like a plateau. 8.
Polymerase chain reaction (PCR) is a method of replicating a specific section of DNA for analysis. In this process the DNA is denatured with heat at 94°C which separates the strands and allows for a primer to be annealed to the strand once the temperature cools to 58°C. Primers are molecules that target specific DNA sequences. dNTPs is then synthesized to the DNA strand by Taq polymerase once the solution is heated to 72°C to extend the
These micelles are imperative for their polar negativity, which causes a pull towards the positive pole. The molecules that are hydrophobic (water hating) will tend to aggregate with the micelle, while those that are hydrophilic (water loving) will move fairly quickly through the solution. The key parameters for this technique are pH, surfactant concentration, any additives and the polymer coatings that are used on the capillary wall. Capillary zone electrophoresis (CZE) This is the most commonly used capillary electrophoresis method of the six being discussed.
Nucleotide excision repair is an important DNA repair system that corrects UV induced genetic damages. One example of such damage is the formation of a covalent bond between two adjacent thymine bases on a DNA strand (thymine dimers). This causes a bulk in the DNA strand and disrupts DNA replication. The process includes the proteins UvrA, UvrB and UvrD as well as DNA polymerase and DNA ligase.
IONIC EXCHANGE CHROMATOGRAPHY This is a form of affinity chromatography that allows for the separation of product and contaminants through the use of specific electrical charges produced by the individual molecules. Resins are applied to depending on the compound one wishes to remove ie if your compound is negatively or positively charged. This system is specifically used after one of the three previously stated methods of separation and an ultrafiltration/diafiltrtion step as it is costly and therefore is not viable for use with large volumes of compound. It is mainly used for the removal of any remaining components from the media and including cell fragments, chemical agents and lipid components. (Duong et al 2014) Ionic exchange resin use
Acceptable criteria: Resolution of NLT 1.5 from primary peak 7. SYSTEM SUITABILITY THEORETICAL PLATES: A standard solution of 25 µg mL-1of Amoxicillin trihydrate (in triplicate) was prepared and same was injected, then the system suitability parameters were calculated. Theoretical plates per meter
Sample preparation and measurement A buffer or detergent or other chemical should not be used unless it will not mask the signal of protein. In addition, compound that could be absorbed in the desired region (190 - 250 nm) should not be used. However, Protein solution should contain only chemicals (with its lowest concentrations) that maintain protein stability. Pure protein should be used if possible, hence any additional peptide or protein will interfere with the CD signal.
Most of the systems vary to include ratios of chloroform, methanol and water. triethylamine, ethanol, hexane, and isopropanol are also common solvents in the mobile phase. Phospholipids migrate to the stationary phase a certain distance on the basis of the composition and affinity for the mobile phase. Identification is based on the delay (Rf), wherein the ratio of the distance moved by the analyte (i.e., the phospholipids) from the origin of the distance moved by the flowing solvent from the origin. Each of the analyte will have its own Rf value under certain circumstances.