A procedure for cloning restriction fragments of DNA as single inserts in yeast artificial chromosomes.
A novel procedure is described for the cloning of partial EcoRI fragments of bovine DNA: it reduces the chance of sequence rearrangements due to multiple insertions (co-cloning) of restriction fragments in the resulting YAC. The DNA to be inserted has been dephosphorylated, whereas the matching ends of the vector, pYAC4, have not. The ligation was essentially complete, the transformation efficiency was close to 19 transformants per ng of vector and the frequency of clones carrying YAC, 60-100 kb in size, was close to 70%. The YACs show segregative and replicative stability.
Dipartimento di Genetica e Microbiologia A. Buzzati-Traverso, Università, Pavia, Italy.
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V
Sgaramella
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Ferretti
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G
Damiani
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Sora
D000818:Animals; D002417:Cattle; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular; D011088:DNA Ligases; D004274:DNA, Recombinant / Q000235:genetics* / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D015151:Immunoblotting; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014170:Transformation, Genetic / Q000502:physiology
D004274:DNA, Recombinant; D011088:DNA Ligases