Isolation and characterization of microsatellite loci for Rhododendron shanii (Ericaceae)
Poly(A) mRNA from the total RNA was purified using oligo(dT) beads. Following purification, mRNA was fragmented into small pieces using divalent cations. The first cDNA strand was synthesized using random hexamer primers for reverse transcription with cleaved mRNA fragments serving as templates. The second strand cDNA was synthesized using DNA polymerase I (Qiagen), incubated with RNase H (Invitrogen), dNTPs and buffer. Following end repair, a single A was added to the blunt end, and then adaptors were added to the purified double stranded cDNA fragments. The products (150C300 bp) are purified and enriched with PCR (15 cycles) to create the final cDNA library.