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KAI-OLAF NETZER
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. Use blunt forceps to transfer the filter to a tray
forthe subsequentwashes. Immediatelyrinsethefilterwith wash buffer 1.
Note: Take care that the filter does not dry.
11. Wash three times for 5 minutes with wash buffer 1 at room temperature.
12. Wash 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
45°C. Between the buffer changes, check the radioactivity remaining in
the center of the blot, where specific signal is expected, in comparison
with the margins of the blot, when background signal is expected.
Note: The washes at 42°C containing decreased cation concentration represent stringency washes. Ifhigher stringency is required, increased time (30
minutes) or decreased salt concentration (0.1x SSC) may be used. The
washes can be carried out in glass trays kept in shaking water baths. Alternatively, washes can be carried out in plastic boxes on a rocker.
14. After sufficient washing, briefly blot the filter on a sheet of 3MM paper.
Seal the damp filter in a plastic bag or plastic wrap.
Note: The filter should not be dried completely if it is to be reused. Probe
cannot be stripperl from the filter if the filter had been dried.
15. Autoradiography of the filter: place the blot (wrapped in plastic seal,
RNA side up) on the bottom of the X-ray cassette, then lay the film
on top. For convenience, the intensifying screen can be affixed to
the cover of the cassette. Close the cassette. Expose at -70°C for one
day up to two weeks.
Note: is convenient to place two films on top of the blot. The film on top can
be developed earlier to check the autoradiograph while the other film is still
exposed to the blot. One film should be taped to the blot so that it cannot be
displaced.
16. When removing the cassette from the freezer, allow sufficient time (30
minutes to one hour) for temperature equilibration. In a dark room,
remove the film from the cassette and develop in an automatic Xray processor.
KAI-OLAF NETZER
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. Use blunt forceps to transfer the filter to a tray
forthe subsequentwashes. Immediatelyrinsethefilterwith wash buffer 1.
Note: Take care that the filter does not dry.
11. Wash three times for 5 minutes with wash buffer 1 at room temperature.
12. Wash 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
45°C. Between the buffer changes, check the radioactivity remaining in
the center of the blot, where specific signal is expected, in comparison
with the margins of the blot, when background signal is expected.
Note: The washes at 42°C containing decreased cation concentration represent stringency washes. Ifhigher stringency is required, increased time (30
minutes) or decreased salt concentration (0.1x SSC) may be used. The
washes can be carried out in glass trays kept in shaking water baths. Alternatively, washes can be carried out in plastic boxes on a rocker.
14. After sufficient washing, briefly blot the filter on a sheet of 3MM paper.
Seal the damp filter in a plastic bag or plastic wrap.
Note: The filter should not be dried completely if it is to be reused. Probe
cannot be stripperl from the filter if the filter had been dried.
15. Autoradiography of the filter: place the blot (wrapped in plastic seal,
RNA side up) on the bottom of the X-ray cassette, then lay the film
on top. For convenience, the intensifying screen can be affixed to
the cover of the cassette. Close the cassette. Expose at -70°C for one
day up to two weeks.
Note: is convenient to place two films on top of the blot. The film on top can
be developed earlier to check the autoradiograph while the other film is still
exposed to the blot. One film should be taped to the blot so that it cannot be
displaced.
16. When removing the cassette from the freezer, allow sufficient time (30
minutes to one hour) for temperature equilibration. In a dark room,
remove the film from the cassette and develop in an automatic Xray processor.