The pooling and superpooling system enables researchers to use PCR for screening a BAC library and identify the specific plate & well containing a BAC clone with a sequence of interest. The screening is done in two separate rounds of PCR on pooled BAC clones (�Round I�and �Round II�).

Each kit is custom built for the researcher and the total number of Superpools in the kit will depend on the total number of BAC clones in the library. Each Superpool will have a corresponding 96-well plate containing all corresponding Plate Pools, Row Pools, Column Pools and Diagonal Pools (called a PRCD plate). For detailed descriptions of these superpools and pools, see pages 11 and 12. The entire resource is aliquoted onto TWO identical working stock, 96-well plate sets (to help reduce the risk of contaminating the entire resource).

The Round I PCR is performed on all the superpools (containing all BAC clones in the library). Each Superpool contains 4,608 individual BAC clones. The results from Round I PCR will identify which Superpool(s) contains BAC clone(s) with the sequence of interest (there may be more than one Superpool identified). The researcher may pursue one or more positive hits found in Round I PCR.

The Round II PCR is performed on the Plate, Row, Column and Diagonal (PRCD) pools for the specific Superpool being investigated. The Round II requires 76 PCR experiments plus controls.
The results from Round II of PCR should allow the researcher to identify the exact plate and well position for one or several positive hits in the particular Superpool under investigation. This allows the
researcher to have identified the specific BAC clone or clones of interest, out of the complete BAC library, that have the sequence where the PCR primers were targeted at.