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Following successful gfp insertion (Fig. 3C) direct induction of the resulting clone copy-number provided, from a single overnight miniculture, quantities of high-quality DNA sufficient for numerous C. elegans transformations (data not shown and Fig. 2D).
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Without such thermostable genetic marker systems, false positive colonies would grow on plates after transformation (data not shown).
In our hands, the naïve, unsupervised equal-width interval binning discretization proved superior to other schemes of data transformation (data not shown).
There was one outlier for HbA1c (changed from 10.5% at baseline to 7.9% at 26 weeks), and the results were similar using a rank transformation (data not shown).
No colonies were seen on selective plates in controls lacking recipient L-forms, donor plasmid, or PEG treatment for transformation (data not shown).
Despite the fact that CSP is also required for maximal transformation of S. mutans, mutants of strain Challis which do not express the challisin also inhibited transformation (data not shown).
Therefore, we suggest that Agrobacterium concentration for genetic transformation should be kept as low as possible (an Agrobacterium concentration corresponding to OD600 = 0.3 was suitable for genetic transformation; data not shown).
Our data show that ectopic expression of PDK1 induces MYC signaling and cellular transformation (data not published); conversely, pharmacologic or genetic removal of PDK1 reduces MYC signaling and alleviates rapamycin resistance.
Nevertheless, VvMYB5bL was still functional despite a growth delay on solid selective medium (6 days) compared to VvMYB5bR recombinant yeasts that were able to develop 4 days after transformation (data not shown).
On one hand, these may act as a barrier to DNA invasion [ 44] and explain why 5632 DNA is resistant to several methylase-sensitive restriction enzymes and to DNA transformation (data not shown).
Control transformations of linearized plasmid DNA alone or PCR amplicons alone did not yield a significant number of transformants (< 20) while the 'complete' transformation contained several hundred transformants (data not shown).
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