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pT7T3-PAC Library Method Overview

Libraries in the pT7T3-PAC vector were constructed in the laboratory of Marcello Bento Soares and M. Fatima Bonaldo, University of Iowa. Briefly, the method was as follows:
  • First strand cDNA synthesis was primed with oligo-dT primer containing a Not I site.
  • Double stranded cDNA was ligated with an EcoR I adaptor, digested with Not I and then cloned directionally into the vector.
  • Each clone in a given library had a different tag sequence located between the Not I site and the polyA tail to identify the library of origin of the clone.

Reference:

Bonaldo MF, Lennon G, Soares MB. Normalization and subtraction: two approaches to facilitate gene discovery. Genome Research 6:791-806, 1996.