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pExpress1 Library Method Overview

Express Genomics, Gaithersburg MD, constructed directionally cloned rat libraries in the pExpress 1 vector using a proprietary method. Briefly, the method overview is as follows:
  • First strand cDNA was synthesized using an oligo-dT primer containing a Not I site and MMLV-reverse transcriptase.
  • Second strand cDNA was synthesized and digested with the Not I restriction enzyme.
  • The double stranded cDNA was size selected (>1.4 kb).
  • The pExpress1 vector was digested with Not I and Eco RV.
  • The double stranded cDNA was directionally cloned into the pExpress1 vector with:
    • the 5' blunt end of the cDNA ligated to the blunt Eco RV site of the vector, thereby destroying this site
    • the 3' end of the cDNA ligated to the Not I site of the vector, thus recreating the Not I site.