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pCR BluntII Topo Vector (Baylor) Library Method Overview

A scalable method for targeted generation of cDNA clones to facilitate recovery of genes absent from the MGC collection was developed in the laboratory of Drs. Richard Gibbs and John McPherson and is outlined below:

Note: This protocol is in the process of being optimized to improve clone recovery.
  • PCR Primer Design
    • The sequence of the targeted gene was found in GenBank. It contained a putative complete ORF, though not necessarily the untranslated region (UTR).
    • Algorithms were developed to pick primers within the 5’ and 3’ UTR. If UTR information was not available, primers were positioned precisely at the beginning and end of the ORF.
    • Gene specific primer pairs were designed based on "Restricted" criteria, the most stringent and, theoretically, the best. If these criteria did not result in primers, "Relaxed" criteria were used, and finally "Forced". The Restricted Criteria were:
      • Complete ORF
      • Starting primer position 80 bp or more from ORF
      • Primer length between 25 and 30 bp
      • GC content between 35% and 75%
      • Tm range between 65oC and 80oC
      • Avoid hair pin structure of three base pairs or more
      • Avoid homology with other sequences
      • Avoid homopolymeric runs of three bases or more
      • Avoid GC clamps

  • RT-PCR
    • mRNA from various human tissues was purchased from a commercial company (Clontech) or total RNA was obtained from Dr. Michael Brownstein (NIMH). In the latter case, mRNA was isolated using µMACS Oligo(dT) MicroBeads from Miltenyi Biotec.
    • The mRNA was converted to double stranded cDNA using the SuperScript III First Strand Synthesis System for RT-PCR (Invitrogen). Briefly:
      • First strand cDNA was synthesized using oligo-(dT)20 primers and SuperScriptTM III reverse transcriptase.
    • The cDNA first strand was amplified by PCR in 96-well plates using Phusion DNA polymerase (MJ Bioworks, now BioRad.)

  • Cloning
    • PCR products were purified using the QuickStep 2, 96-well purification kit (Edge Biosciences).
    • Purified amplicons were cloned into the pCR BluntII TOPO cloning kit (Invitrogen).