19 cDNA Library Screening
307
8. Supernatants are discarded and the inner walls of the tubes are carefully
wiped out using a soft paper.
9. The two pellets derived from the same phage clone are resuspended and
pooled in a total of 4 ml of SM. The milky pellet material is homogenized
by pipetting up and down with a 5 ml pipette.
10. Extract PEG by adding 4 ml CHC13 and vortex for 30 sec; centrifuge at
1,600 g for 15 min at 4°C.
11. The aqueous (top) phase is transferred into a fresh 12 ml polypropylene
tube, and the volume is adjusted with SM to 4 ml precisely. The jelly-like
organic phase and the interphase are discarded.
12. In a 14 ml polyallomer ultracentrifuge tube (Kontron) a CsCl step gradient is prepared containing from bottarn to top: 3 ml CsCl with a density of p=1.7; 1.5 ml (p=l.5); and 1.5 ml (p=1.45).
13. 2 g solid CsCl are dissolved in each of the 4 ml phage suspensions in SM.
14. The phage suspensions are layered onto the CsCl step gradient and centrifuged at 30,000 rpm for 2 hrs at 15°C in a Beckman SW40 rotor or
equivalent.
15. The gradients are carefully removed from the rotor and placed under a
strong lamp. At this point phage particles are clearly visible as a milky
band at the interphase between density zones 1.5 and 1.45. The phage
particle are harvested using an 18G needle punctured through the side
of the tube. V olume is approx. 1.5 ml.
16. The volume of the harvested sample is brought to 6 ml with SM, and
solid CsCl ( usually approx. 3 g) is added until the refractive index of the
solution is precisely 1.380. The volume of the sample is then further
expanded with CsCl solution (0.75g/ml) to a final volume of 9.5 ml.
17. Phage particles are re-centrifugated through a continuous density gradient (e.g. 30,000 rpm in a Beckman Ti75 rotor overnight at 20°C).
18. Phage particles are collected as in step 15 and areready for DNA extraction by standard procedures (Maniatis et al. 1989).
307
8. Supernatants are discarded and the inner walls of the tubes are carefully
wiped out using a soft paper.
9. The two pellets derived from the same phage clone are resuspended and
pooled in a total of 4 ml of SM. The milky pellet material is homogenized
by pipetting up and down with a 5 ml pipette.
10. Extract PEG by adding 4 ml CHC13 and vortex for 30 sec; centrifuge at
1,600 g for 15 min at 4°C.
11. The aqueous (top) phase is transferred into a fresh 12 ml polypropylene
tube, and the volume is adjusted with SM to 4 ml precisely. The jelly-like
organic phase and the interphase are discarded.
12. In a 14 ml polyallomer ultracentrifuge tube (Kontron) a CsCl step gradient is prepared containing from bottarn to top: 3 ml CsCl with a density of p=1.7; 1.5 ml (p=l.5); and 1.5 ml (p=1.45).
13. 2 g solid CsCl are dissolved in each of the 4 ml phage suspensions in SM.
14. The phage suspensions are layered onto the CsCl step gradient and centrifuged at 30,000 rpm for 2 hrs at 15°C in a Beckman SW40 rotor or
equivalent.
15. The gradients are carefully removed from the rotor and placed under a
strong lamp. At this point phage particles are clearly visible as a milky
band at the interphase between density zones 1.5 and 1.45. The phage
particle are harvested using an 18G needle punctured through the side
of the tube. V olume is approx. 1.5 ml.
16. The volume of the harvested sample is brought to 6 ml with SM, and
solid CsCl ( usually approx. 3 g) is added until the refractive index of the
solution is precisely 1.380. The volume of the sample is then further
expanded with CsCl solution (0.75g/ml) to a final volume of 9.5 ml.
17. Phage particles are re-centrifugated through a continuous density gradient (e.g. 30,000 rpm in a Beckman Ti75 rotor overnight at 20°C).
18. Phage particles are collected as in step 15 and areready for DNA extraction by standard procedures (Maniatis et al. 1989).