Simultaneous loading of 200 sample lanes for DNA sequencing on vertical and horizontal, standard and ultrathin gels

We have developed a simple and efficient technique for automated parallel loading of . 200 lanes on a 30 cm-wide gel in automated DNA sequencing, using porous filter materials and an associated manual or robotic system. The samples are loaded onto the teeth of a comb made of the porous material. The comb, with samples, is inserted directly above the straight edge of the polymerized gel. The samples are driven from the comb into the gel by the applied electrical field. A particularly advantageous aspect of this method is the elimination of the thin gel walls separating the sample wells in the standard gel loading technique. The time for sample loading is significantly reduced to a few minutes. The loading technique is applicable to horizontal or vertical systems, with standard or ultrathin gels. In the standard gel casting and loading protocols, a plastic comb is used to form sample wells in the polymerized gel. The plastic comb is removed after polymerization, samples loaded in the wells, electric field applied and the separation process started. Problems with this technique are encountered when dimensions of the sample slots are small, i.e., for widths <2 mm. In systems with high throughput, large numbers of samples per gel are required, and the slot width may be 31.5 mm. When the gel walls separating the slots are very thin (<1 mm) they may become damaged, leading to mixing of different samples, distorted band patterns and unreliable results. The problem is worse with gels <0.5 mm thick. The ‘shark tooth’ technique avoids the problem of thin separating walls, but the manual sequential loading into the small sample slots remains tedious, time consuming and prone to errors. We have developed a simple and efficient technique for automated parallel loading of . 200 lanes on gel in automated DNA sequencing, using combs made of porous filter materials and an associated manual or robotic system. The gel used was 5% Hydrolink, 1· TBE. Casting of the gel and sequencing protocols were as described in ref. (1). To prepare the straight and smooth edge of the gel on which the samples are loaded, a polyester spacer with a straight and smooth edge, of the thickness of the gel, Figure 1. Sample block (A), with the teeth of the porous comb (B) inserted in the sample pockets (C) for drawing the samples in the comb. Optionally, a plastic strip (D; polyester, width 20 mm, thickness 0.3 mm) is fixed to the comb above the teeth as indicated, to improve its handling.