10 Hybridization Methods (Southern and Northern Blotting)
143
8. Allow transfer to proceed for 12-16 hours. Make sure that there is
enough 20x SSC in the reservoir (approximately 3 1).
9. After blotting, carefully disassemble the blotting apparatus. Transfer
the membrane together with the gel to a dry filter paper, gel side up.
Mark the position of the gel and the wells on the fllter membrane
by using a soft pencil. Peel off the gel.
10. Wash the fllter membrane briefly in 20x SSC to remove any traces of
agarose.
Note: It is important to wash the filter in 20x SSC. Washing with lower ionic
strength buffer may remove RNA from the fllter.
11. Place the fllter membrane briefly on a dry sheet of 3MM paper to blot
dry.
12. Fix the RNA to the filter membrane. Expose the nylon filter RNA side
down to UV light for 3-5 minutes as described above (see Chap. 10).
13. The filter is now ready for hybridization or can be stored at 4 oc. Nylon
filters should be sealed in plastic bags.
Subprotocol 6
Hybridization of RNA Blots
Usually, RNA blots are hybridized at 42°C in hybridization buffers containing 50o/o formamide. Higher temperatures increase the rate of RNA degradation and are therefore not recommended. The following is a standard
protocol.
Materials
• DNA or RNA probe labeled to high specific activity (approximately 10 9
cpm/J.tg) with e.g. random primed labeling (DNA), or in vitro transcription (RNA). For protocols see Chap. 9.
• RNA containing Northern filter
• 20x SSPE (3.6 M NaCl, 0.2 M NaH2P04, 0.02 M EDTA, adjust the pH to 7.5
with 10 N NaOH [approximately 6 ml/1])
143
8. Allow transfer to proceed for 12-16 hours. Make sure that there is
enough 20x SSC in the reservoir (approximately 3 1).
9. After blotting, carefully disassemble the blotting apparatus. Transfer
the membrane together with the gel to a dry filter paper, gel side up.
Mark the position of the gel and the wells on the fllter membrane
by using a soft pencil. Peel off the gel.
10. Wash the fllter membrane briefly in 20x SSC to remove any traces of
agarose.
Note: It is important to wash the filter in 20x SSC. Washing with lower ionic
strength buffer may remove RNA from the fllter.
11. Place the fllter membrane briefly on a dry sheet of 3MM paper to blot
dry.
12. Fix the RNA to the filter membrane. Expose the nylon filter RNA side
down to UV light for 3-5 minutes as described above (see Chap. 10).
13. The filter is now ready for hybridization or can be stored at 4 oc. Nylon
filters should be sealed in plastic bags.
Subprotocol 6
Hybridization of RNA Blots
Usually, RNA blots are hybridized at 42°C in hybridization buffers containing 50o/o formamide. Higher temperatures increase the rate of RNA degradation and are therefore not recommended. The following is a standard
protocol.
Materials
• DNA or RNA probe labeled to high specific activity (approximately 10 9
cpm/J.tg) with e.g. random primed labeling (DNA), or in vitro transcription (RNA). For protocols see Chap. 9.
• RNA containing Northern filter
• 20x SSPE (3.6 M NaCl, 0.2 M NaH2P04, 0.02 M EDTA, adjust the pH to 7.5
with 10 N NaOH [approximately 6 ml/1])