BME100 Lab 1_nigel

BME100 Lab 1_nigel - BME 100 Laboratory 1 Dissociation of...

Info iconThis preview shows pages 1–4. Sign up to view the full content.

View Full Document Right Arrow Icon
BME 100 Laboratory 1 Dissociation of Double-stranded Polynucleotides Nigel Chou October 1, 2007 Instructor: Fan Yuan
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
BME 100 Lab 1 1. Introduction Polynucleotides, such as DNA and RNA, are important in biology, and the association and dissociation of individual strands, held together by hydrogen bonding, play an important part in their function. It is thus instructive to study the thermodynamic properties of this process. In this lab, we investigate the dissociation of a synthetic polynucleotide, polyinosinic acid-polycytidinic acid (polyI-polyC), as a function of temperature. The degree of dissociation is analyzed using the phenomenon of hyperchromicity, which results in dissociated strands have a higher light absorption than associated-strands, due to the lack of stacking interactions in single strands.[1] Hence a higher absorption corresponds to a greater degree of dissociation of polyI-polyC. A spectrophotometer is used measure the absorbance of polyI-polyC as the temperature of the solution increases from 25°C to 90°C, which is then correlated with the fraction of double-stranded polyI-polyC and plotted. The melting temperature, T m, is determined from the plot. Using this information, plots of Δ r H and Δ r S against temperature are made and the results analyzed to determine the thermodynamic characteristics of the system. 2. Materials and Methods Materials 1) 0.1 mM solution of polyI-polyC 2) Deionised water (for Blank solution) 3) 2 cuvettes (h = 1cm) 4) Spectrophotometer 5) Water bath maintained at 90°C over a hot plate 6) 1.5ml eppendorf tube (to hold sample in the water bath) Methods First, the spectrophotometer was set-up by setting the wavelength set at 251nm and specifying the blank and sample positions. 1 ml of polyI-polyC solution and 1 ml of blank solution were placed into two separate cuvettes and loaded into the spectrophotometer in the correct orientation, with the transparent sides facing the sensor. The spectrophotometer chamber was set to the desired temperature, starting with 25°C followed by 7 other readings ranging from 45°C to 60°C. After each temperature adjustment, we ensured that the temperature display had stabilized at the desired temperature before the measurement was taken. The spectrophotometer directly calculated and displayed the difference between the absorbance of blank solution and that of polyI-polyC solution, which were printed and recorded. The cuvette holding the polyI-polyC solution was then transferred to an eppendorf tube and placed in a 90°C water bath for 5 min, before being removed and immediately returned the 1
Background image of page 2
BME 100 Lab 1 spectrometer for a final measurement. For this step, the spectrophotometer’s temperature setting was held at the temperature of the previous measurement (60°C). Derivation of Equations
Background image of page 3

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Image of page 4
This is the end of the preview. Sign up to access the rest of the document.

This lab report was uploaded on 04/09/2008 for the course BME 100 taught by Professor Yuan during the Fall '07 term at Duke.

Page1 / 12

BME100 Lab 1_nigel - BME 100 Laboratory 1 Dissociation of...

This preview shows document pages 1 - 4. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online