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Example sentences for: pyub
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The pYUB178 plasmid and the λ phage library of pYUB178::H37Rv cosmids were generously provided by Dr. William Jacobs, Albert Einstein University, New York, NY [ 20 ] . The cosmid library contains 30-50 kb fragments of M. tuberculosis genomic DNA generated by partial Sau 3A digestion cloned into Bcl I-digested pYUB178.
The PCR product was used as a probe for colony blots to find cosmids in the E. coli (pYUB178::H37Rv) library carrying at least a portion of the cosmid integrated in the sur2 clone.
Probing colony blots of the E. coli (pYUB178::H37Rv) library with the 0.8 kb PCR fragment led to the isolation of a 4 kb plasmid.
Portions of the E. coli (pYUB178::H37Rv) cosmid library were plated on LB agar containing 50 μg kanamycin/mL, and colony blots were performed with the ECL Direct Nucleic Acid Labeling & Detection System (Amersham) according to manufacturer's instructions.
To enrich for clones with increased survival in human macrophages, the library was passed through THP-1 macrophages as shown schematically in Figure 1. THP-1 is a human monocyte-derived macrophage cell line [ 21 ] and wild-type M. smegmatis bacteria are rapidly killed by THP-1 macrophages [ 22 ] . After three rounds of enrichment, the resulting clones were analyzed individually by Southern blot to evaluate the number of different clones present as previously described [ 20 ] . Out of the 3000 colonies recovered, genomic DNAs from the bacteria of 90 randomly chosen colonies were digested with Pst I and hybridized with the pYUB178 vector.
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