Standard Pulsed Field Gel Running Conditions

There exists a large and probably confusing variety of pulsed-field gel equipment at present. The basic device, OFAGE (orthogonal field alternate gradient electrophoresis, ref. 13, see also Chapter 4), has been modified and the second generation of equipment includes FIGE (field inversion gradient electrophoresis, ref. 2, see also Chapter 1), CHEF (contour-clamped homogeneous electric field, ref. 14, see also Chapter 2), TAFE (transverse alternating field electrophoresis, ref. 15, see also...

Reptation Theory of Pulsed Electrophoresis and Trapping Electrophoresis

Jean Louis Viovy and Anne Dominique Defontaines In the history of the still-young technique of pulsed electrophoresis experimental, theoretical, and numerical progress are increasingly intermingled. The first key to this field can be traced to theoretical letters of Lerman and Frisch and Lumpkin and Zimm (1,2), who remarked that long flexible chains like chromosomal DNA cannot migrate in gels as random coils, and suggested a reptative-like motion in which chains thread their way among fibers...

Catrin Pritchard and Margit Burmeister

Pulsed-field gel electrophoresis (PFGE) has the capacity to fractionate large fragments of DNA up to thousands of kilobases in size. This aspect of the technique has been exploited for constructing longrange restricdon maps of chromosomes from many different species including humans (see Chapters 14, 15, and 18). Besides its use for analytical purposes, PFGE has also been used as a preparative tool. Intact DNA obtained from preparative PFGE gels has been used for cloning into yeast artificial...

Protoplast Isolation for Several Dicotyledonous Plants

We usually use young plant material from the greenhouse, however, it is also possible to use in vitro grown plants. The use of high intensity lights should be avoided, because often the plants are stressed and accumulate anthocyanins and starch in their tissue. If you find that your tissue contains excessive starch, place the plants in the dark for 1-2 d before protoplast isolation. 2. Feathering leaves along the veins reduces the amount of debris significantly and makes removal of undigested...

Nancy C Stellwagen

DNA molecules less than 20 kilobase (kb) pairs in size are efficiently fractionated by unidirectional electrophoresis in agarose gels (1). The variation of DNA mobility with gel concentration can be described by the Ogston mechanism of pore size distribution, if the observed mobilities are first extrapolated to zero electric field strength (2). Therefore, sieving of the macromolecules by the matrix appears to be the dominant mechanism of mol wt separation in this size range. DNA molecules...

Beyond Biased Reptation

The biased reptation model rather well describes the mobility of long DNA in agarose and acrylamide, and under a wide variety of pulsed-field conditions. A notable exception is FIGE, for which biased reptation leads to qualitatively wrong predictions. Experiments investigating electrophoretic motions on a molecular scale and computer simulations were also developed in order to better understand the mechanisms of PFGE. These studies, presented in more detail in several sections of this volume,...

Preparation of Yeast Size Markers Method 1

Streak out yeast on YPD plates and incubate at 30 C for 2 d. Yeast live about 3-4 wk on an agar plate and should be stored permanently as a glycerol stock at -80 C. 2. Grow a single large colony overnight at 30 C in 50 mL of YPD. Fig. 1. Panel A shows ethidium bromide stained mouse genomic DNA separated by PFGE in the 0.05-1.5 Mb size range. Lane 1 uncut control, Lane 2 S. cerevisiae YP80, Lane 3 BssHII (sample damaged on loading), Lane 4 BssHII +Notl, Lane 5 BssHII + Mlul (sample damaged on...

Variation Among Species

The genomic complexity, and the number and size of the chromosomes (karyotype, which includes the mitochondrial DNA), differ widely when various species of protozoa are compared (see below)-, even a comparison of closely related species reveals significant differences in genome content and organization (for examples of kinetoplastidae 5-8,14-19 T. cruzi 20 for examples of Eimeria 21 Pneumocystis cannu 22 Theileria 23 Plasmodium 24-26 Gtardia, 27-29 ). A karyotype that is characteristic for each...

InGel Dnadna Hybridization

As the concentration of both agarose and salt in a glycerol-free agarose gel increases, the curling that occurs during air-drying of the gel increases. Although addition of glycerol prevents curling, glycerol is not added for the first drying (Step 1 in Section 3.2.) because of a resultant increase in the nonspecific background retention of 32P-labeled probe in the gel. The first washing of the gel (Step 1 in Section 3.2.) minimizes curling by removing salt. Without the salt, gels less...