Итак, был получен якобы очищенный вирус и из этого материала была извлечена вся РНК. Затем делали вот что.
cDNA library construction 55 ng of purified viral RNA was used in construction of a random primed and oligo-dT primed cDNA library, using the SuperScript Choice System for cDNA synthesis (Invitrogen). Linkers were ligated following cDNA synthesis. The cDNA synthesis products were visualized on agarose gels, revealing the anticipated low-yield smear. To produce sufficient cDNA for cloning, the cDNA product was size fractionated on a lowmelting point preparative agarose gel, followed by PCR amplification using a single PCR primer specific to the linkers. This yielded sufficient material for cloning. Size selected cDNAs were ligated into the pCR4-TOPO TA cloning vector from Invitrogen. cDNAs were then transformed by electroporation into DH10B T1 cells (Invitrogen), plated on 22 cm 2 agar plates with the appropriate antibiotic and grown for 16 hours at 37°C. Colonies were picked into 384-well Axygen culture blocks containing 2 × YT media and grown in a shaking incubator for 18 hours at 37°C. Cells were lysed and DNA purified using standard laboratory procedures.
1. Произвели обратную транскрипцию РНК в кДНК, используя случайные праймеры и праймеры для poly(A)-хвоста, специфичного для мРНК.( Collapse )