18 cDNA Libraries
287
Pellet the cells by centrifugation at 2000 x g for 10 min. Resuspend the
cells in half the original volume of cold 10 mM MgS04• The cells should be
stored at 4°C until use (within 24 h).
3. Incubate the ligation mix for 30 min at - 20°C, thaw at room temperature
and then add to the highest efficiency packaging extract immediately
after thawing the latter. Incubate at room temperature for 2 h.
4. Dilute the packaging mix with phage dilution buffer (100 mM NaCl, 20
mM Tris-HCl (pH.7.4), 10 mM MgS04) to a final volume of 2 ml, add a
few drops of chloroform, gently mix (-3 s) and place on ice. The library is
now ready for titering.
5. The library should be kept at 4°C until plating. Packaged bacteriophage
are unstable in vitro and will suffer a significant drop in titer if not plated
within the next 24- 48 hours.
6. Order of magnitude dilutions are prepared for titering. Aliquots of diluted extract are adsorbed to 200 ~-tl of host cells for 15-20 minutes at
37°C, added to 7 ml of melted top agar (cooled to below 50°C), and
poured onto dry, pre-warmed agar plates.
7. The plates are incubated at 37°C overnight. Cloning efficiency is expressed as the number recombinants/~-tg cDNA and is calculated using
the following formula: (number of recombinant plaques) x ( dilution factor) I (number of /-lg cDNA).
8. Plate the remainder of the library at a dilution adequate to yield -SO x 10 4
plaques/130 mm plate.
References
Aviv Hand Leder P (1972) Purification ofbiologically active globin messenger RNA by
chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sei USA 69:1408
1412
Gubler U and Hoffman BJ (1983) A simple and very efficient method for generating
cDNA libraries. Gene 25:263 269
Huynh TV, Young RA and Davis RW (1984) Construction and screening cDNA libraries
in lambda gtlO and lambda gt11. In DNA Cloning, Vol1:49 78. IRL Press, Oxford
Patanjali SR, Parimoa S and Weissman SM (1991). Construction of a uniform-abundance (normalized) cDNA library. Proc Natl Acad Sei USA 88:1943 1947
287
Pellet the cells by centrifugation at 2000 x g for 10 min. Resuspend the
cells in half the original volume of cold 10 mM MgS04• The cells should be
stored at 4°C until use (within 24 h).
3. Incubate the ligation mix for 30 min at - 20°C, thaw at room temperature
and then add to the highest efficiency packaging extract immediately
after thawing the latter. Incubate at room temperature for 2 h.
4. Dilute the packaging mix with phage dilution buffer (100 mM NaCl, 20
mM Tris-HCl (pH.7.4), 10 mM MgS04) to a final volume of 2 ml, add a
few drops of chloroform, gently mix (-3 s) and place on ice. The library is
now ready for titering.
5. The library should be kept at 4°C until plating. Packaged bacteriophage
are unstable in vitro and will suffer a significant drop in titer if not plated
within the next 24- 48 hours.
6. Order of magnitude dilutions are prepared for titering. Aliquots of diluted extract are adsorbed to 200 ~-tl of host cells for 15-20 minutes at
37°C, added to 7 ml of melted top agar (cooled to below 50°C), and
poured onto dry, pre-warmed agar plates.
7. The plates are incubated at 37°C overnight. Cloning efficiency is expressed as the number recombinants/~-tg cDNA and is calculated using
the following formula: (number of recombinant plaques) x ( dilution factor) I (number of /-lg cDNA).
8. Plate the remainder of the library at a dilution adequate to yield -SO x 10 4
plaques/130 mm plate.
References
Aviv Hand Leder P (1972) Purification ofbiologically active globin messenger RNA by
chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sei USA 69:1408
1412
Gubler U and Hoffman BJ (1983) A simple and very efficient method for generating
cDNA libraries. Gene 25:263 269
Huynh TV, Young RA and Davis RW (1984) Construction and screening cDNA libraries
in lambda gtlO and lambda gt11. In DNA Cloning, Vol1:49 78. IRL Press, Oxford
Patanjali SR, Parimoa S and Weissman SM (1991). Construction of a uniform-abundance (normalized) cDNA library. Proc Natl Acad Sei USA 88:1943 1947