PMC

Stability study of BAC clones. DNA samples were isolated from 6 single BAC clones derived from 1st and 5th cultures (0-generation cells and 100-generation cells) and digested with HindIII. M 1 Kb ladder DNA marker+λDNA/HindIII; a–f 6 single BAC clones; I 0-generation cells II, 100-generation cells.

Analysis of the cotton BAC clones. (a) Ethidium bromide-stained agarose gel showing 13 BAC clones digested with NotI and separated by PFGE. The size marker is the midrange PFGE marker II and its sizes are indicated in the margin. (b) An autoradiograph of the same gel hybridized with total cotton genomic DNA. (c) Distribution of insert sizes based on the analysis of 103 clones randomly selected from the BAC library.

The scheme representations of BAC pooling and PCR-based screening strategy. “→” indicated the direction of BAC pooling. Step I, one column pool was mixed with each 384-well column and 16 clones in the same column (gray dot) were combined into a column pool. Step II, One super pool was mixed with each column pool in the same raw and the column pools in same raw (black dot) were mixed. The entire BAC library of 123, 648 clones on 322 384-well plates was organized into 322 super pools. “⇢” indicated the direction of three rounds of PCR-based screening. Round I, screening against 322 Super pools. Round II, screening against the individual row of column pools. Round III, screening against the individual column of particular plate(s).