INTERPRETATION OF HYBRIDISATION RESULTS

The filter is labelled at the top left hand corner (NIH Dros). 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 the filter.

There is one large filter supplied. Sheet (i) shows a typical 22 x 22 cm membrane with 9,216 spotted clones. Each filter is divided into 6 panels (indicated by ,, .....). The panels are numbered from left to right and eight 96-well plates are gridded, in duplicate, in each panel. Within each panel, numbers 1-12 correspond to the 12 columns and letters A-H are the 8 rows of the eight 96-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 1536 clones (96 x 8 x 2). Table (ii) shows the order of the 8 plates (in duplicate) within each panel on the filter. Panel contains plates 1-8, contains 9-16 and so on. This plate order has been maintained on all the membranes, e.g.

	NIH Dros, Panel						NIH Dros, Panel
	plates	3	5	6	8		plates	11	13	14	16
		2	7	3	4			10	15	11	12
		6	4	8	5			14	12	16	13
		2	1	7	1			10	9	15	9	....etc.

(Please note that the NIH Dros. filters have the following missing plates: 1,2,25,38,39,43,47 and 48)

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-H) and column (1-12) 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:

(1st plate)	(2nd plate)	(3rd plate)	(4th plate)
(5th plate)	(6th plate)	(7th plate)	(8th plate)
Use either table (ii) or table (iii) to identify which plate number is giving the positive signal.

5. Record the identity of putative positives by following the convention
   PLATE-ROW and COLUMN (e.g. 16-D8). When requesting this clone please select the library NIH Drosophila cDNA and enter the clone as 16-D8. Please note that in publications or database entries the correct name of this clone would be bs16d8.