High EDTA concentrations cause entrapment of small DNA molecules in the compression zone of pulsed field gels, resulting in smaller than expected insert sizes in YACs prepared from size selected DNA

Many procedures for the preparation of yeast artificial chromosome (YAC) libraries use size selection of insert DNA by pulsed field gel electrophoresis (PFGE) to remove small molecules and increase the average size of clones (1). In most cases, the compression zone containing the focused partial digestion products is used for ligation to artificial chromosome arms. In some cases, particularly in the construction plant YAC libraries, electrophoresis is used to remove short molecules from the DNA plugs themselves, which are then retrieved from the gel and used for ligation and transformation (eg. 2, 3). Both approaches have been used successfully, although the average insert size with the latter procedure is shorter. Here we demonstrate that the method used to stop the partial digestion reaction, and in particular the level of EDTA in the DNA plugs before loading them on a gel, drastically influences the size distribution of molecules compressed in the limiting mobility band. This is reflected in the insert sizes of YAC clones prepared from the focused DNA. Chromosomal soybean DNA was embedded in agarose plugs and prepared for restriction digestion as described (5). Partial digestion with EcoRl was carried out using 0.4 to 4 units of enzyme per 40 fi\ plug in a final volume of 100 /d. The reactions were quenched by adding 100 /d of 100 mM EDTA (to produce a final EDTA concentration of 50 mM) or 100 ^1 of 500 mM EDTA as in (3). The reaction mixtures were incubated at 37 °C with 1 mg/ml proteinase K. The plugs were then loaded on a 0.9% LMP agarose gel and electrophoresed for 12 h at 200 V, 3 s switch interval, in 0.25 xTBE. Partially digested DNA was recovered from the compression zone and ligated to dephosphorylated YAC4 vector. The ligation mixture was used directly for transformation of yeast strain AB138O as described (5).