3.2 METAL ENJEKSİYON KALIPLARINDA YOLLUK TASARIMI
3.2.7 Yolluk Tasarım Kriterleri
Os produtos gerados nas reações de PCR foram analisados eletroforeticamente em agarose, como descrito por Sambrook et al. (1989). O programa de ciclagem (denominado DOWNTM) foi o mesmo para ambos os pares de primers ISBA (rDNA16S), mat (alkB primer degenerado) e matg (alkB primer específico), como descrito pela metodologia de Santos (2006).
Para amostras de DNA metagenômicos e DNA genômico de consórcios bacterianos foi utilizado o seguinte protocolo de PCR:
tampão de PCR10X (Biosystem), 2 µL de primer foward (20 pmol), 2 µL primer reverse (20 pmol), 1 µL DNTPs e 2,5 U de Taq polimerase (Biosystem).
Para amostras de DNA genômico dos isolados bacterianos crescidos dos microcosmos que foram extraídas pelo método de “extração por fervura” foi utilizado o seguinte protocolo de PCR:
x 5µL ácidos nucléicos dos consórcios, 1 µL de 2,5 mM MgCl2, 2
µL de tampão de PCR 10X (Biosystem), 2 µL de primer foward (20 pmol), 2 µL primer reverse (20 pmol), 1 µL DNTPs e 2,5 U de Taq (Biosystem) e/ou GoTaq® Flexi DNA Polymerase (PROMEGA).
Após a reação de PCR com mDNA, as amostras foram analisadas em gel de agarose a 1,8%.
Depois da avaliação com gel de agarose, os amplicons foram analisados pela técnica de PCR-DGGE, utilizando o aparelho DGGE-1001 (C.B.S., Scientific Company). As análises foram feitas em poliacrilamida a 8% durante 16 horas a 80 V e 22 mA, com gradiente de desnaturação uréia-formamida de 40% a 60%. Em cada canaleta do DGGE foram aplicados 10 ȝl das reações de PCR. Os géis foram corados com nitrato de prata a 0,2% e fotodocumentados.
48
4. RESULTADOS E DISCUSSÃO
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