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RNA was reverse-transcribed to first strand cDNA using SuperScriptII reverse-transcriptase and tagged oligo-dT primer which contains several restriction sites including a NotI site: gactagttctagatcgcgatcgcgaGCGGCCGCccttttttttttttttt. Second strand DNA was synthesized by E. coli DNA polymerase I in combination with E. coli RNAse H and E. coli DNA ligase. Double stranded cDNA was ligated with SalI adapter. These cDNAs were cloned into the SalI/NotI site of pBluescript KS+ and transformed into E. coli Electromax DH10B by electroporation.
Nucleotide
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