INTERPRETATION OF HYBRIDISATION RESULTS

The filters are labelled at the top left hand corner (AG1 and AG2). This label serves as an orientation mark. The enclosed sheets [i, and tables (ii) and (iii)] provide detailed information about the layout of clones on each filter.

There is one large filter and one small filter supplied. 21 plates have been gridded onto the small filter which is a half of the size of the big filter. As this is the first time that we have provided this new size of filter we would appreciate if you could let us know if the use of this filter creates any problems.

Sheet (i) shows a typical 22 x 22 cm membrane with 36,864 spotted clones. Each filter is divided into 6 panels (indicated by 1,2 ....6). The panels are numbered from left to right and eight 384-well plates are gridded, in duplicate, in each panel. Within each panel, numbers 1-24 correspond to the 24 columns and letters A-P are the 16 rows of the eight 384-well plates in the panel. Each dot represents a clone from a single microtitre plate well. A group of 16 dots corresponds to a 4x4 array of duplicate clones from the 8 microtitre plates (8 x 2). Thus each panel consists of 6144 clones (384 x 8 x 2). Table (ii) shows the order of the 8 plates (in duplicate) within each panel on the filter. Panel 1 contains plates 1-8, panel 2 contains 9-16 and so on. This plate order has been maintained on all the membranes, e.g.

The interpretation of positive signal(s) can be done by following the procedure given below:

1. Identify the membrane on which the positive signal is located (in duplicate).

2. Use the label position to orientate the filter and identify the panel in which the positive clones are located.

3. Work out the microtitre co-ordinates of the 4 x 4 array by referring to the row (A-P) and column (1-24) designations.

4. Within the identified 4 x 4 array determine which plate number contains the positive clone.

Check that any two signals fall in one of the following patterns:

Use either table (ii) or table (iii) to identify which plate number is giving the positive signal.

5. Record the coordinates of putative positives by following the convention

PLATE-ROW and COLUMN (e.g. 16-L15). When requesting this clone it should be preceded by the short library name (e.g. AG16-L15) and clearly identified as the result of Geneservice filter screening.

30^th Apr. 1997 Version 2.1 (2AG)