To test the software sensitivity, a previously identified heterozygous mutant (G1642A in Wx-D1) was used to mix with non-mutant DNA to form mutant:non-mutant ratios of 1 0, 1 1, 1 2, 1 3, 1 4 and 1 5 so that the mutant allele fractions in the pooled DNA were 1/2, 1/4, 1/6, 1/8, 1/10 and 1/12.
Finally, it would be of interest to mix the mutant and isogenic control Δ3 4-hiPS cells in different relative proportions prior to neuronal differentiation to recapitulate the mosaic mutant and WT MECP2 expression pattern that exists in RTT girls for downstream applications.
Thus, local building codes are these ever-changing and constantly-swelling mutant books made up of whichever aspects of these different codes local governments choose to mix together.
We further assayed the ability of ctPan2 mutants to bind ctPan3 PKC by mixing separately purified proteins and analyzing whether they co‐elute on size exclusion chromatography.
The purified proteins, MBP-μ1/2/3-CTD (0.86 mg) or α398-σ (0.73 mg) and MBP-BST2CD/MBP-VpuCD (0.73 mg) or their mutants, were mixed to a final volume of 500 µl.
We then performed error-prone PCR on this mix, and assayed 435 mutants to measure the fraction functional (see Additional file 2).
Similarly, the μ1-CTD-truncated AP1 (3.4 mg) and MBP-VpuCD (1.2 mg, wt or the ELV/AAA mutant) were mixed to a final volume of 500 µl.
