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We explored the feasibility of rapid screening of gDNA library with PCR technique. We adopted porcine alpha-1,3GT cDNA fragment as the probe, used primers synthesized by the specific sequence on cDNA to carry out alpha-1,3GT gene screening of porcine gDNA library by combining PCR and in situ plaque hybridization, and then performed enzymatic digestion, Southern blot, sequencing, and fluorescence in situ hybridization for location. After having finished one-time hybridization and one-time PCR, we obtained 7 positive monoclones with very strong signals, and each insert length of them was over 8 kb, including the third intron. Moreover, 3 tested clones among them contain the third and fourth exons according to sequencing results. FISH mapped the inserts of the 3 clones to pig chromosome 1q2.10-q2.11. PCR could be applicable to rapid screening of DNA library and this approach is much simpler compared to conventional in situ plaque hybridization alone.