138
KAI-OLAF NETZER
Note: W ear gloves when working with radioactive probes, and work behind
plexiglass shielding. Work according to your laboratory's safety rules. Dispose of radioactive waste properly.
8. Incubate at 65°C for 12-16 hours in a shakingwaterbath or between two
glass plates in an oven.
9. After hybridization, open the bag at one corner and squeeze out the
hybridization mix into a 15 or 50 ml Falcon tube. Take care not to spill
radioactive solution.
Note: The hybridization solution may be reused once.
10. Open the bag completely. U se blunt forceps to transfer the filter to a tray
for the subsequent washes. Immediately rinse the filter with wash buffer
1.
Note: Take care that the fllter does not dry.
11. W ash three times for 5 minutes with wash buffer 1 at room temperature.
12. W ash two times for 5 minutes with wash buffer 2 at room temperature.
13. Washone to three times for 15 minutes with preheated wash buffer 2 at
65°C. Between the buffer changes, use a Geigercounter to check the
radioactivity remaining in the center of the blot, where specific signal
is, expected in comparison with the margins of the blot, where background signal is expected.
Note: The washes at 65°C containing decreased SSC concentration represent stringent washes. If higher stringency is required, increased time (30
minutes) or decreased salt concentration (0.1x SSC) may be used. Washes
can be carried out in plastic boxes with tightly sealing lids in shaking water
baths. Alternatively, washes can be carried out in a hybridization oven using
roHer bottles or a rocker.
14. After sufficient washing, brietly lay the filter on a sheet of 3MM paper to
blot dry. Seal the damp fllter in a thin walled plastic bag or plastic wrap.
Note: The filter should not be dried completely if rehybridization is intended. Probe cannot be stripped from the filter if the filter had been dried.
15. Autoradiography of the filter: place the blot (wrapped in plastic seal,
DNA side up) on the bottom of the X-ray cassette, then lay the film
on top. The intensifying screen can be conveniently affixed to the cover
of the cassette. Close the cassette. Expose at -70°C for overnight or up to
two weeks.
KAI-OLAF NETZER
Note: W ear gloves when working with radioactive probes, and work behind
plexiglass shielding. Work according to your laboratory's safety rules. Dispose of radioactive waste properly.
8. Incubate at 65°C for 12-16 hours in a shakingwaterbath or between two
glass plates in an oven.
9. After hybridization, open the bag at one corner and squeeze out the
hybridization mix into a 15 or 50 ml Falcon tube. Take care not to spill
radioactive solution.
Note: The hybridization solution may be reused once.
10. Open the bag completely. U se blunt forceps to transfer the filter to a tray
for the subsequent washes. Immediately rinse the filter with wash buffer
1.
Note: Take care that the fllter does not dry.
11. W ash three times for 5 minutes with wash buffer 1 at room temperature.
12. W ash two times for 5 minutes with wash buffer 2 at room temperature.
13. Washone to three times for 15 minutes with preheated wash buffer 2 at
65°C. Between the buffer changes, use a Geigercounter to check the
radioactivity remaining in the center of the blot, where specific signal
is, expected in comparison with the margins of the blot, where background signal is expected.
Note: The washes at 65°C containing decreased SSC concentration represent stringent washes. If higher stringency is required, increased time (30
minutes) or decreased salt concentration (0.1x SSC) may be used. Washes
can be carried out in plastic boxes with tightly sealing lids in shaking water
baths. Alternatively, washes can be carried out in a hybridization oven using
roHer bottles or a rocker.
14. After sufficient washing, brietly lay the filter on a sheet of 3MM paper to
blot dry. Seal the damp fllter in a thin walled plastic bag or plastic wrap.
Note: The filter should not be dried completely if rehybridization is intended. Probe cannot be stripped from the filter if the filter had been dried.
15. Autoradiography of the filter: place the blot (wrapped in plastic seal,
DNA side up) on the bottom of the X-ray cassette, then lay the film
on top. The intensifying screen can be conveniently affixed to the cover
of the cassette. Close the cassette. Expose at -70°C for overnight or up to
two weeks.