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An electrophoretic technique called pulsed eld gel electrophoresis has been speci cally developed for separations in the mass range 200 3000 kb (Example 55) 581 Physical Basis of Pulsed Field Gel Electrophoresis All the electrophoretic techniques we have so far encountered in this chapter use a constant and unidirectional electrical eld However, in pulsed eld gel electrophoresis, the direction of the eld is varied continuously during electrophoresis (Figure 537) This is achieved by alternately turning on and off the current or pulsing the electrical eld in short time intervals called the pulse time, tp Intact chromosomal DNA or large fragments of chromosomal DNA are obtained by rst lysing cells in an agarose matrix by treatment with a detergent or with enzymes such as lysozyme This treatment often generates fragments of chromosomal DNA due to mechanical shearing.
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Pyrolysis Pyrolysis is a technique normally used to analyze nonvolatile organic compounds such as wood, paper, or polymers by GC/MS. In pyrolysis the sample is heated rapidly to 750 C or higher in order to thermally decompose it. A high temperature such as this is su cient to break the polymer backbone, forming smaller, more volatile fragments. By examining these fragments, it is sometimes possible to deduce the structure of the polymer chain. Some polymers unzip during pyrolysis to yield only the original monomer. This technique is used frequently to examine materials for the presence of additives such as plasticizers, antioxidants, ame retardants, ultraviolet (UV) stabilizers, or sizing treatments applied to cloth samples. Solids Probe Direct solids probe analysis is a volatilization technique that places the sample under vacuum near the mass spectrometer s ion source. The sample s temperature can either be raised to a preset maximum or it can be heated gradually, in a temperatureprogrammed fashion. During this time, the molecules that volatilize from the sample continuously enter the mass spectrometer s source and are ionized much in the same fashion as described above with GC/MS. The disadvantage of this technique is that there is no separation step. This is yet another way to analyze nonvolatile samples using mass spectrometry.
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and P ( s ) may be interpreted as the local wavelength of the oscillation divided by 27r. The quantities a,p, and y are usually referred to as Courant-Snyder parameters collectively; from now on, the function p will be referred to as the amplitude function. So the general solution to the equation of motion can be written as
Large fragments may also be generated, if required, by digestion with unusual restriction enzymes recognizing rarely-occurring 8-nucleotide restriction sites (eg Asc I, Not I) Slices of agarose containing large DNA molecules are then loaded on 06 15% agarose gels and electrophoresis is initiated An array of electrodes is arranged around the gel such that the direction of electrophoresis may be continuously varied (see next section) Pulses of electricity are passed through these electrodes for times in the range 01 s to 90 min before changing the direction of the eld and passing pulses of electricity through the eld in the new direction (Figure 537) The direction of electrophoresis of DNA is constantly alternated between the two electric eld directions as the molecules reorientate themselves through a reorientation angle.
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