Notes

1. Glass bead DNA isolation kits are commercially available from Bio 101 (La Jolla, CA). The GeneClean II kit is apparently able to recover DNA from Tns-borate as well as Tris-acetate agarose gels.

2. Microinjection DNA is dissolved in a buffer of 10 mM Tris-HCl, pH 7.4,0.1-0.25 mM EDTA. Higher concentrations of EDTA and low concentrations of MgCl2 are toxic to eggs.

3. Many manipulations of DNA and RNA leave the nucleic acid in a solution containing unwanted salts, nucleotides, or radioactive moieties. In the majority of cases, ethanol precipitation will not entirely remove these or will add further salts. For this reason, Sephadex gel-exclusion chromatography is often employed to purify DNA fragments. Sephadex is a bead formed, crosslinked dextran. The crosslinking is carefully controlled, giving rise to pores of a particular size. Each bead therefore can be considered to consist of a network of holes, all having identical size and shape. A molecule passing down a Sephadex column will pass through a volume that is dependent on the size of the molecule A molecule that is too large to enter a pore will only pass through the volume of the column not occupied by the beads. A small molecule (such as a solute molecule) will be small enough to enter the pores and, therefore, the molecule will pass through a much larger volume and so take a longer time to pass down the column. Since the size of the pore can be controlled, a range of gel-exclusion (or gel-filtration) matrices are produced. The size of a molecule that can just enter the pore is called the exclusion limit (i.e., larger molecules are excluded). Pharmacia (Uppsala Sweden), for example, makes eight Sephadex gel-filtration matrices, G-10, G-15, G-25, G-50, G-75, G-100, G-150 and G-200, in a range of grades (superfine, fine, medium, and coarse). Their exclusion limits are, respectively, 700,1500, 5000,10,000,50,000,100,000,150,000, and 200,000 daltons Note that these limits are calculated for Dextrans. Molecules of intermediate size will fractionate according to their mol wt. For example, G-50 will fractionate in the range 500-10,000 daltons. Therefore, a mixture of salts and DNA will fractionate cleanly. The DNA will be excluded from the pores and so will pass straight through the column. Salts and nucleotides will be small enough to enter the pores and so will be retained on the column. One further advantage of this method is that the sample does not become appreciably diluted.

4. DNA fragments are injected at a concentration of 1-5 (ig/mL. Excessively high concentrations of DNA will kill eggs (2). It is estimated that around 1-2 pL of DNA solution are introduced into the pronucleus by microinjection. Thus, depending on the size, around 500 copies of the DNA fragment are introduced. Some workers make dilutions of the injection DNA stock (e.g., 1, 2.5, and 5 (ag/mL) and rotate between these different solutions during a microinjection session as the pipets are changed.

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