INTERPRETATION OF HYBRIDISATION RESULTS

INTERPRETATION OF HYBRIDISATION RESULTS

 

Each filter is labelled at the top left hand corner (Mouse PAC1, Mouse PAC2 ..........Mouse PAC7). This label serves as an orientation mark. The enclosed sheets (i, ii - v and vi) provide detailed information about the layout of clones on each of 7 filters.

 

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). Sheet (ii) shows the order of the 8 plates (in duplicate) within each panel of filters Mouse PAC1 and Mouse PAC2. Panel 1 contains the first 8 plates 337 - 344, panel 2 contains the next 8 plates, 345 - 352 and so on.
This plate order has been maintained on all the membranes, e.g.

 

mouse_PAC_RPC121plates

 

Sheet (iii) shows the filters Mouse PAC3 and Mouse PAC4

Sheet (iv) shows the filters Mouse PAC5 and Mouse PAC6

Sheet (v) shows the filter Mouse PAC7

 

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:

    mouse_PAC_RPC121plates2

  5. Record the identity of putative positives by following the convention
    PLATE-ROW and COLUMN (e.g. 564-L15).

 

Sheet (vi) allows for an alternative method of interpretation

 

Version 1.0
14th October 1997
 

NB: On filter PAC7 only 1 plate is gridded on panel 5 and none on panel 6 as no more plates were available.
          plates 174-176, 284, 285 and 311 do not exist