19 cDNA Library Screening
299
Second, the dry fllter membrane is carefully placed onto the bacterial
lawn starting from the center of the plate towards the rim. From the
time when the membrane has become completely wet (color turns
gray) wait another 30 seconds (1.5 minutes in case of a second lift).
Meanwhile, mark the position of the membrane on the plate by three
asymmetrically placed needle sticks. Carefully remove the fllter from
the plate and place contact side up on a clean sheet of Whatman
3MM. Step 4 is repeated for all of the plates before proceeding to
the next step.
5. Float membranes contact side up on top of denaturation solution for 5
minutes.
6. Neutralize for 5 minutes.
7. Washin 2 x SSC for 5 minutes.
8. Place membranes on a fresh sheet ofWhatman 3MM and wrap in aluminum foil together with the supporting paper.
9. Bake for 2 hrs at 80°C in a vacuum oven.
10. Briefly prewet membranes in 6 x SSC then prewash in prewashing solution at 68°C for at least 1 hr. Use a tray placed in a shaking water bath.
11. For prehybridization, 25 filter membranes are transferred into a preserving jar ( 90 mm at least) filled with 50 ml ofhybridization solution.
Prehybridization is performed at 68°C for 2 hrs.
12. Foreach ml of the hybridization solution 2 times10 5 to 1xl0 6 cpm of
double-stranded probe are denatured by boiling in a water bath for 10
minutes followed by rapid cooling on ice.
13. The denatured probe is then immediately added to the prehybridization mix.
14. Hybridization is performed at 68°C overnight in the sealed preserving
jar under constant agitation to prevent adherence of filter membranes
to each other.
15. After hybridization, filter membranes are washed 3 times for 1 hr in 2 x
SSC, 0.01 o/o SDS at room temperature.
16. Nitrocellulose membranes are then mounted while still damp (keep
from drying!) onto sheets of stiff paper or X-ray fllm and the needle
marks are labeled with radioactive ink.
299
Second, the dry fllter membrane is carefully placed onto the bacterial
lawn starting from the center of the plate towards the rim. From the
time when the membrane has become completely wet (color turns
gray) wait another 30 seconds (1.5 minutes in case of a second lift).
Meanwhile, mark the position of the membrane on the plate by three
asymmetrically placed needle sticks. Carefully remove the fllter from
the plate and place contact side up on a clean sheet of Whatman
3MM. Step 4 is repeated for all of the plates before proceeding to
the next step.
5. Float membranes contact side up on top of denaturation solution for 5
minutes.
6. Neutralize for 5 minutes.
7. Washin 2 x SSC for 5 minutes.
8. Place membranes on a fresh sheet ofWhatman 3MM and wrap in aluminum foil together with the supporting paper.
9. Bake for 2 hrs at 80°C in a vacuum oven.
10. Briefly prewet membranes in 6 x SSC then prewash in prewashing solution at 68°C for at least 1 hr. Use a tray placed in a shaking water bath.
11. For prehybridization, 25 filter membranes are transferred into a preserving jar ( 90 mm at least) filled with 50 ml ofhybridization solution.
Prehybridization is performed at 68°C for 2 hrs.
12. Foreach ml of the hybridization solution 2 times10 5 to 1xl0 6 cpm of
double-stranded probe are denatured by boiling in a water bath for 10
minutes followed by rapid cooling on ice.
13. The denatured probe is then immediately added to the prehybridization mix.
14. Hybridization is performed at 68°C overnight in the sealed preserving
jar under constant agitation to prevent adherence of filter membranes
to each other.
15. After hybridization, filter membranes are washed 3 times for 1 hr in 2 x
SSC, 0.01 o/o SDS at room temperature.
16. Nitrocellulose membranes are then mounted while still damp (keep
from drying!) onto sheets of stiff paper or X-ray fllm and the needle
marks are labeled with radioactive ink.