19 cDNA Library Screening
301
Procedure
Preparation of Sepharose-coupled bacterial lysate
1. 2 x 7.5 ml LB media are each inoculated with a single colony ofthe lysogenic temperature-sensitive E. coli strain and grown overnight at
32°C.
2. The overnight cultures are diluted 100-fold in LB and grown at 32°C to
an OD600 of 0.5.
3. Phage expression is induced by incubation at 45°C for 15 minute.
4. Further incubate cultures at 39°C for approx. 2 hrs.
5. To test phage content and readiness for harvesting, CHCh-tests are performed: 3 drops of CHCh are added to a 1 ml test sample of culture and
the mixture is then incubated at 37°C. A control aliquot of culture to
which no CHCh had been added is incubated in parallel. After 5 minutes of incubation the test sample is compared to the control. If the
tested cell suspension has cleared upon addition of CHCh the bacteria
are fully packed with phage and justabout to lyse. At this stage the culture is ready for harvesting.
6. Harvest bacteria by centrifugation at 4,000 x g for 10 minutes at 4 °C.
7. Cell pellets from both original 7.5 ml cultures are resuspended separately in cold coupling buffer and respun.
Note: coupling buffer must be cold (4°C), otherwise premature Iysis of cells
will occur.
8. Cells are resuspended in each 5 ml cold coupling buffer and pooled.
9. Sonicate cell suspension until the increased viscosity caused by Iiberation of nucleic acids disappears.
10. Couple the bacteriallysate to the previously prepared CNBr-activated
Sepharose 4B overnight at 4°C.
11. Recover the coupled material by centrifgation at 3,000 x g for 5 minutes.
12. Wash pellet once by resuspension in coupling buffer and recentrifugation.
13. For saturation of unoccupied reactive groups of the CNBr-activated Sepharose the pellet is resuspended in cold (4°C) saturation buffer and
incubated for 1 hr at 4°C on a rotating wheel.
301
Procedure
Preparation of Sepharose-coupled bacterial lysate
1. 2 x 7.5 ml LB media are each inoculated with a single colony ofthe lysogenic temperature-sensitive E. coli strain and grown overnight at
32°C.
2. The overnight cultures are diluted 100-fold in LB and grown at 32°C to
an OD600 of 0.5.
3. Phage expression is induced by incubation at 45°C for 15 minute.
4. Further incubate cultures at 39°C for approx. 2 hrs.
5. To test phage content and readiness for harvesting, CHCh-tests are performed: 3 drops of CHCh are added to a 1 ml test sample of culture and
the mixture is then incubated at 37°C. A control aliquot of culture to
which no CHCh had been added is incubated in parallel. After 5 minutes of incubation the test sample is compared to the control. If the
tested cell suspension has cleared upon addition of CHCh the bacteria
are fully packed with phage and justabout to lyse. At this stage the culture is ready for harvesting.
6. Harvest bacteria by centrifugation at 4,000 x g for 10 minutes at 4 °C.
7. Cell pellets from both original 7.5 ml cultures are resuspended separately in cold coupling buffer and respun.
Note: coupling buffer must be cold (4°C), otherwise premature Iysis of cells
will occur.
8. Cells are resuspended in each 5 ml cold coupling buffer and pooled.
9. Sonicate cell suspension until the increased viscosity caused by Iiberation of nucleic acids disappears.
10. Couple the bacteriallysate to the previously prepared CNBr-activated
Sepharose 4B overnight at 4°C.
11. Recover the coupled material by centrifgation at 3,000 x g for 5 minutes.
12. Wash pellet once by resuspension in coupling buffer and recentrifugation.
13. For saturation of unoccupied reactive groups of the CNBr-activated Sepharose the pellet is resuspended in cold (4°C) saturation buffer and
incubated for 1 hr at 4°C on a rotating wheel.