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4 x 4 Grid Clones, THREE Fields

How to identify an address.

4 x 4 THREE Field Picture Diagram

The membrane is divided into 3 fields (click above link for diagram). Each field contains 384 squares. The 384 squares represent the row and column identification of the BAC. Within each square there are 16 positions where 8 clones are spotted in duplicate (see diagram). The pattern of the spotted clones will generate the plate address of the BAC. To identify your clone, please follow the directions below.

The most complicated part about identifying a clone address is that consecutive plates are not spotted into each field. The 384 well plates are spotted onto the membrane with plates 1-3 spotted into fields 1-3 respectively (duplication pattern 1, see diagram). Since there is a total of 3 fields on the membrane, the cycle will continue with the next three consecutive plates (plates 4 through 6) again being spotted into fields 1 through 3 respectively, but in a different duplication pattern (duplication pattern 2, see diagram). This gridding cycle will continue until all the plates have been spotted.

1. The library name and filter number is used to orient the membrane. Place the membrane with the label facing up and on the right-hand side as shown in the diagram. (The colonies are on the same side of the filter as the label)
2. Identify the field number of the hybridizing colonies. The spacing of colonies is slightly wider between the fields.
3. Identify the well location (I have included a grid to help and identify the well position (e.g. L18)
4. Identify the plate number. This is accomplished by determining the orientation of the duplicate spots (duplication pattern in the diagram) and referring to the table inside each field in the figure.

Example: If you have horizontal spots, they could either be duplication position 4 or 8 (from the duplication pattern). They are distinguished by the closeness of the spots and position in the pattern. Assume it is position 4 in field 3. Read down the table in field 3 of the diagram to pos4 and read the plate number as 12. The library plate number is 12. Once the plate number is determined, identify the well location either by using the library plate grid (see link for this) or counting the rows and columns.
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4 x 4 THREE Field Picture Diagram
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