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The data from low quality amplification were removed from HRM analysis.
The melting curves of specimens that have a good quality amplification curve and a crossing point >35 cycles are reviewed by technical supervisor and microbiologist.
For each hybrid, 42 polymorphic markers were selected that distinguished the parental lines and which gave high quality amplification in prior analyses.
As controls for RNA quality, amplification reactions were performed using pairs of primers specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Egidy et al, 2000).
Among 58 primer pairs producing good quality amplification product, 9 returned product sizes larger than expected, which could be due to simultaneous amplification of an intron during the PCR.
Thirty-one DNA samples from different west Eurasian countries (Germany, France, Poland, Bosnia-Herzegovina, Romania, Switzerland, Italy, Austria, Turkey, Kazakhstan, Denmark, Portugal, Spain, and the Russian Federation) were subjected to entire mtDNA control region sequencing according to high quality amplification and sequencing procedures [6].
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Of the 14 primers tested, 12 were polymorphic and two were monomorphic with high-quality amplification (Table 2).
Consistent and high-quality amplification of all tested Sebacina isolates occurred for eight primer sets (Table 2).
Of the 37 primer pairs tested, we identified eight polymorphic and four monomorphic loci with high-quality amplification (Table 1).
However, our present results showed that the use of Bouin's solution as a fixative was also associated with low-quality amplification.
The majority of S. latifolia EST-SSRs generated high-quality amplification products, suggesting that ESTs are ideally suited for specific primer design.
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