Two parts of the DNA melter require you to do some design work. There are instructions below for assembling your DNA melter. Don't worry about that - in part 2, you will make many improvements to your machine that will make it sing like Elvis. You will probably notice some shortcomings your instrument as you work. After you have a melting curve, you will use nonlinear regression to fit thermodynamic parameters to your data and do a little planning for part 2 of this lab.
Melting point measure software#
Computer software for recording the voltages has already been written for you. This involves recording the temperature and fluorescence voltage as you heat a sample of DNA + dye from room temperature to about 95☌ and then let it cool back down. The goal of part 1 is to produce at least one reasonable quality melting curve. When you get it all put together, the instrument will produce two voltages: one related to the sample temperature and another that depends on the amount of fluorescence coming out of the sample. The instrument includes a resistance temperature detector (RTD) to measure the temperature of the sample holder, a blue LED to excite the sample, a Peltier device that will heat the sample, optical filters, a photodiode for detecting fluorescent emission, a high-gain transimpedance amplifier to convert photocurrent to voltage, and a computer data acquisition system to record the voltages that come out of the instrument. Even though this first version of the instrument has some shortcomings, it will give you a good idea of how all of the parts work together to make melting curves. In this part of the lab, you will construct a crude version of a temperature-cycling fluorometer and measure a DNA melting curve or two. Note that unlike boiling point, the melting point is relatively insensitive to pressure and no pressure correction needs to be made.Normalized DNA melting curve. temperature changing only about 2 ☌/min) until you obtain two consistent values.
Then carry out at least two further careful determinations (by heating more gently, i.e. If, however, you are looking for confirmation of the purity of a substance and therefore know roughly what temperature your sample will melt at, this rough estimation may not be needed. It is standard practice (in order to make the most effective use of time)to carry out a rapid melting point determination initially (by heating rapidly) to establish an approximate melting point. Place the sample in your melting point apparatus or Thiele tube and start heating. Place the sample in a capillary tube and seal the end. Familiarise yourself with the requirements of your particular melting point determining method. Care needs to be taken to ensure that heating does not happen too fast Method The tube is filled with a suitable liquid, most commonly an oil and the side arm is heated.Īs the liquid is heated, convection causes the liquid to circulate around the system distributing the heat. The sample in a capillary tube is held next to the bulb of a thermometer by eg a rubber band and placed in the ‘main’, straight part of the tube. The Thiele tube is basically a set tube with a side loop (see diagram). They come in at around £600.Ī cheaper alternative, that with care can still produce accurate results, is the Thiele tube. The one problem with the melting point apparatus described above is that of cost. The most commonly a heated metal block such as a Mel-Temp apparatus Thiele Tube The sample is loaded in a sealed capillary tube and the temperature gradually raised by means of an internal metal block. This is the most common piece of apparatus for determining melting point (as shown in the picture at the top of the page). There are a few ways of doing this Melting point apparatus The general method is to heat a sample indirectly by placing the prepared sample (either packed in a glass capillary or on a glass cover slip) in or on a heated medium and observing it, and the temperature, closely until melting is complete. Pure samples usually have sharp melting points, for example 149.5-150☌ or 189-190☌ impure samples of the same compounds melt at lower temperatures and over a wider range, for example 145-148☌ or 186-189☌. You can find out more about melting points below. 
Determining the melting point of a compound is one way to test if the substance is pure and is often used to test samples made from organic synthesis (eg of aspirin or paracetomol).
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