By Ivan Lefkovits, Benvenuto Pernis
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Additional info for Immunological Methods. Volume IV
2. PRE adapters have two functions: they are used (1) to convert a staggered restriction end into a 3' homopolymer tail and (2) to provide cDNA molecules with both a chi site and a staggered restriction end suitable for ligating to vector molecules. Each function requires a pair of partially complementary partner oligonucleotides. PRE adapters can Earn SI XI ^—"ERI K. ERI 9ar? \ CI857 Sam 5 cosR n i n Fig. 1. Structure of Xjac and Xecc. XB, the structure of which is shown, highlighting the areas altered in the new vectors.
The technology described here might seem much more complicated than other protocols for generating cDNA libraries, and certainly the prepara tion of the vector does involve more steps than, say, the simple cleavage of XgtlO with EcoRl. However, once the oligonucleotide reagents are in hand, it takes only 2 days to prepare sufficient vector for many libraries, and the insertion of cDNA sequences actually involves fewer steps than do most comparable alternatives. Nonetheless, it must be admitted that, if the goal is to prepare a basic cDNA library to be screened only by plaque hybridization with defined probes, the insertion of double-stranded cDNA into XgtlO and selection on hfl E.
Vol. 65, p. 347. Academic Press, New York. , and Honjo, T. (1986). Nature (London) 324, 70. Patterson, T. , and Dean, M. (1987). Nucleic Acids Res. 15, 6298. Perry, R. , Kelley, D. , and Greenberg, J. R. (1972). Biochim. Biophys. Acta 262, 220. , and Wu, R. (1980). In "Methods in Enzymology" (L. Grossman and K. ), Vol. 65, p. 43. Academic Press, New York. Ryffel, G. , and McCarthy, B. J. (1975). Biochemistry 14, 1379. , Fritsch, E. , and Maniatis, T. (1989). "Molecular Cloning: A Laboratory Manual," 2nd Ed.
Immunological Methods. Volume IV by Ivan Lefkovits, Benvenuto Pernis