Four hundred super pools of 20 groups were PCR screened.
Super pools of twenty five 384-well plates and two-dimension matrix pools of the BAC library were prepared for marker screening.
The "super pool" format of 2008, when Australia, New Zealand and England were placed in the same group, was deemed a success and will be considered again.
According to the proposed pooling strategy, the required number of super pools for a typical 3× coverage BAC library with a 100 Kb average insert size is estimated for several important genomes (Table 2).
The samples for each run were equimolarly pooled (super-pools of respectively 23, 24 and 47 samples) and subsequently each of the three super-pools was size fractioned using a 1% (w/v) agarose gel.
With the isolated DNAs quantified by PicoGreen, the purified insert DNAs were portioned out to create an equal molar super-pool of all 11 component fosmid pools, as well as a set of 3 nested equal molar super-pools of 8, 4, and 2 small fosmid pools.
Twenty super-pools of BAC clones were generated for each of the ten 96-well plates by combining the wells from twelve vertical rows and eight horizontal columns.
A cod BAC library consisting of 92,000 clones with average insert size of 125 kb was screened for globin genes by PCR using gene specific primers (Table 2) on pools and super-pools of BAC clones.
Modified MT-PCR is performed in 20 μL containing 10 μL of the supplied 2× Biomix (Biomix kit, Bioline), 2 μL freezing stock from the super pool, 5 μM of each primer (forward and reverse) multiplexed up to 50 markers and 4 mM MgCl2.
The second step of the screening relied on serial dilutions of the super-pools to inoculate 50 bacteria from the positive per well in a 96 deep-well plate.
To screen the BAC library using each of 119 pairs of comparative oligo primer pairs, the diluted DNA from each well of the super pool plates was used as a DNA template.
