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 NIH dros hybrid no 1,NIH dros hybrid no 2, .....NIH dros hybrid no 6). 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 NIH dros hybrid no 1 contains plates 1-8, NIH dros hybrid no 2 contains 9-16 and so on. This plate order has been maintained on all the membranes, e.g.

NIH Dros, Panel NIH dros hybrid no 1 NIH Dros, Panel NIH dros hybrid no 2
plates 03 05 06 08 plates 11 13 14 16
  02 07 03 04   10 15 11 12
  06 04 08 05   14 12 16 13
  02 01 07 01   10 09 15 09


(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:

NIH dros hybrid plate 1 NIH dros hybrid plate 2 NIH dros hybrid plate 3 NIH dros hybrid plate 4
(1st plate) (2nd plate) (3rd plate) (4th plate)
NIH dros hybrid plate 5 NIH dros hybrid plate 6 NIH dros hybrid plate 7 NIH dros hybrid plate 8
(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.