Interpretation of hybridisation results


Each filter is labelled at the top left hand corner (FUGU1, FUGU2,.......FUGU5). This label also serves as an orientation mark. The enclosed sheets (i, ii-iv and v) provide detailed information about the layout of clones on each of 5 filters.

Sheet (i) shows a typical 22 x 22 cm membrane with 36,864 spotted clones. Each large 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 spots (384 x 8 x 2). The figure at the top of sheet (ii) shows the order of the 8 plates (in duplicate) within each panel of filters Fugu1-Fugu4. 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.

Fugu cosmid1

 

Sheet (iii) shows the filter (FUGU5). Since only 3 plates are left for this last filter a small membrane (7.8 x 11.9 cm) has been used to grid the remaining 3 plates.

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:


Fugu cosmid2


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

 

PLATE-ROW and COLUMN (e.g. 164-L15).

 

Sheet (iv) allows for an alternative method of interpretation