The proportion of mutant plaques was expected to be directly related to the ratio of mutant to wild-type genomes in the primary mixed plaque.
The procedure used by Besaratinia et al. [ 11] to amplify cII mutant plaques was adapted here for lacZ mutant plaques.
Blue mutant plaques were identified against a red background on a lightbox, and re-plated to verify as true mutants.
Comparison of expected and observed mutant count when the number of each mutant plaque was controlled for in each NGS library.
In each case 218 bp dsDNA substrates were used, mutant-containing plaques were identified by PCR analysis of 18 individual plaques with a primer complementary to the His6 tag, and purified mutants were identified after re-plating and re-testing (Table 3).
Similar proportions of mutant-containing plaques were observed using substrates that generate a 717 bp in-frame deletion in Giles gene 20, and increasing the amount of 200 bp dsDNA substrate did not substantially alter the proportion of mixed plaques detected by flanking PCR (Table 1).
To recover the 402 bp gene 20 deletion mutant, ten mixed plaques were picked and re-plated and single plaques tested by PCR; at least one pure mutant was identified in seven of these, although at greatly varying frequencies (1/15, 2/15, 3/16, 10/15, 2/17, 2/24, 2/15; 19.8% average).
"The blue plaques are extraordinary.
In mutant epidermis, electron-dense desmosomal plaques were significantly reduced in number particularly between basal cells and between basal and suprabasal cells (Fig. 1E).
Plaques were formed by all mutants expressing only one VPg and 2C with R55W, and they were visible at 3 days post transfection.
