Individual DNA concentrations were normalized to 40 ng/ul and then used to prepare 8 fold pooling plates for TILLING screens.
Screening of genomic DNA from the TILLING library was conducted on two fold pools in consideration of the high mutation density associated with this hexaploid wheat EMS-mutagenised population.
In the 1-dimensional format, each sample is present once in a single eight-fold pool per 96-well plate.
HRM of these fragments efficiently identifies the 6-fold pool containing the mutant fish and a second round of HRM of these six fish identifies the mutant individual, which was confirmed by Sanger sequencing and recovered from cryopreserved sperm.
Once a mutation was revealed, the eight-fold pool (samples: 1 8) was remixed into eight discrete pools consisting of two individuals each (samples: 1&2, 3&4, 5&6, 7&8, 1&3, 2&4, 5&7, and 6&8).
For screening the mutant library, M2 DNAs were first subjected to a four-fold pooling, resulting in 752 pooled samples contained in eight 96-well microtiter plates.
With 4-fold pooling all of the mutant bands were detected, as they were with the 8-fold pools, whereas with 12-fold pooling only a subset of the mutant bands were seen on the TILLING gel.
Lowering the pooling fold (for example, 4-fold pooling) may increase the sensitivity of mutation detection in sorghum.
DNA was then pooled using a one-dimensional eight-fold pooling strategy.
Eight-fold pools of genomic DNA from leaf tissues of M2 plants were used for TILLING.
HRM analysis of EMS-mutagenized maize was conducted with 5-fold pooling [ 10].
