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Positive transformants were determined by DNA sequencing and transformed into strain Rosetta2 (DE3) (Novagen).
After validating the mitochondrial localization of PaMT1-his in T4 and T5, copper levels in mitochondria of these transformants were determined (Fig. 4D).
The expression levels of Smmob3 and pro11 in the RNAi transformants were determined by quantitative real-time PCR.
The QRDRs of several independent transformants were sequenced to confirm the presence of the same mutation in the donor DNA and MICs of these transformants were determined.
Positive transformants were determined by PCR, using primers M13F (5' GTA AAA CGA CGG CCA G 3') and M13R (5' CAG GAA ACA GCT ATG AC 3') provided by the manufacturer.
In order to identify the mutations that had been transferred to 1974T3 and R6T2, the genome sequences of the parent mutant 1974M1 and of both transformants were determined by 454 pyrosequencing.
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For each transformation, the proportion of Tr colonies among 50 EmR PmR transformants was determined.
A standard curve was generated using N-acetylglucosamine as a standard, and the chitinase activity of the transformants was determined (Reissig et al. 1995).
The xylanase activity of the transformants was determined by the xylan-overlay assay.
The insertion copy number of transformants was determined by their resistance to G418 and transformants with the same copy number were selected.
The MIC values of clarithromycin of 10 randomly selected resistant transformants was determined by agar dilution and were identical to those of donor strains.
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