10 Hybridization Methods (Southern and Northern Blotting)
139
Note: One can save exposure time by placing two films on top of the blot.
The film on top can be developed earlier to check the autoradiograph while
the other film - which should be taped to the blot - is still exposed.
Subprotocol 4
Preparation of RNA Dot Blots
RNA dot blots are used to determine the presence or absence of speciflc
transcripts. In comparison with standard samples, the amount of target
RNA may also be quantifled. It is not possible, however, to determine
the sizes of mRNA transcripts. To achieve this, Northern blotting is the
method of choice.
Materials
Use RNase-free reagents or DEPC-treated reagents only. Wear gloves when
working with RNA.
• Formaldehyde, 37o/o solution (handle in a fume hood)
• Formamide (handle in a fume hood)
• 20x SSC (3M NaCl, 0.3 M sodium acetate)
• Denaturing solution (500 J..ll formamide, 162 )ll formaldehyde [37o/o solution], 100 )ll 20x SSC)
• Membrane filter. The use of nylon membranes is recommended due to
their superior handling properties.
Procedure
1. Cut a piece of filter membrane to the appropriate size. Mark the locations RNA blot
where the RNA will be spotted with a soft pencil. A one cm-grid may be hybridization
helpful.
2. Soak the membrane in 20x SSC.
3. Denature 10-20 )lg total RNA by incubation for 5 minutes at 65°C in the
following solution:
139
Note: One can save exposure time by placing two films on top of the blot.
The film on top can be developed earlier to check the autoradiograph while
the other film - which should be taped to the blot - is still exposed.
Subprotocol 4
Preparation of RNA Dot Blots
RNA dot blots are used to determine the presence or absence of speciflc
transcripts. In comparison with standard samples, the amount of target
RNA may also be quantifled. It is not possible, however, to determine
the sizes of mRNA transcripts. To achieve this, Northern blotting is the
method of choice.
Materials
Use RNase-free reagents or DEPC-treated reagents only. Wear gloves when
working with RNA.
• Formaldehyde, 37o/o solution (handle in a fume hood)
• Formamide (handle in a fume hood)
• 20x SSC (3M NaCl, 0.3 M sodium acetate)
• Denaturing solution (500 J..ll formamide, 162 )ll formaldehyde [37o/o solution], 100 )ll 20x SSC)
• Membrane filter. The use of nylon membranes is recommended due to
their superior handling properties.
Procedure
1. Cut a piece of filter membrane to the appropriate size. Mark the locations RNA blot
where the RNA will be spotted with a soft pencil. A one cm-grid may be hybridization
helpful.
2. Soak the membrane in 20x SSC.
3. Denature 10-20 )lg total RNA by incubation for 5 minutes at 65°C in the
following solution: