10 Hybridization Methods (Southern and Northern Blotting)
141
Changes in size or Ievel of expression of mRNA may be the cause for altered
amount of protein product or altered biological activity.
Materials
Use RNase-free reagents or DEPC-treated reagents only. Wear gloves when
working with RNA.
• Formaldehyde/agarosegel containing size separated RNA
• Formaldehyde, 37% solution (handle in a fume hood)
• Formamide (handle in a fume hood)
• 1 Ox MO PS ( 0.2 M 3- [N-morpholino] propanesulfonic acid, 0.5 M sodium
acetate pH 7.0, 0.01 M EDTA)
• Loading buffer (SO% glycerol, 0.1 mg/ml bromophenol blue, 0.1 mg/ml
ethidium bromide [optional, if direct UV-visualization of RNA is desired], in 1x MOPS buffer)
• Water bath (50°C)
• 20x SSC (3M NaCl, 0.3 M sodium citrate)
• Membrane filter. The use of nylon membranes is recommended due to
their superior handling properties.
Procedure
1. Run a formaldehyde/agarose gel according to the following protocol with ln advance
10-20 J..tg total RNA per lane or 0.5-1 J..tg poly(A)+ RNA, and photograph
the gel on a UV -transilluminator alongside a ruler (see Chap. 4).
a. Incubate total RNA (10-20 J..tg) for 5 minutes at 65°C in the following
solution:
Total RNA (10-20 J..Lg)
Formamide
lOx MOPS buffer
Formaldehyde (37%)
6.0 J.ll
12.5 J.ll
2.5 J.ll
4.0 J.ll
141
Changes in size or Ievel of expression of mRNA may be the cause for altered
amount of protein product or altered biological activity.
Materials
Use RNase-free reagents or DEPC-treated reagents only. Wear gloves when
working with RNA.
• Formaldehyde/agarosegel containing size separated RNA
• Formaldehyde, 37% solution (handle in a fume hood)
• Formamide (handle in a fume hood)
• 1 Ox MO PS ( 0.2 M 3- [N-morpholino] propanesulfonic acid, 0.5 M sodium
acetate pH 7.0, 0.01 M EDTA)
• Loading buffer (SO% glycerol, 0.1 mg/ml bromophenol blue, 0.1 mg/ml
ethidium bromide [optional, if direct UV-visualization of RNA is desired], in 1x MOPS buffer)
• Water bath (50°C)
• 20x SSC (3M NaCl, 0.3 M sodium citrate)
• Membrane filter. The use of nylon membranes is recommended due to
their superior handling properties.
Procedure
1. Run a formaldehyde/agarose gel according to the following protocol with ln advance
10-20 J..tg total RNA per lane or 0.5-1 J..tg poly(A)+ RNA, and photograph
the gel on a UV -transilluminator alongside a ruler (see Chap. 4).
a. Incubate total RNA (10-20 J..tg) for 5 minutes at 65°C in the following
solution:
Total RNA (10-20 J..Lg)
Formamide
lOx MOPS buffer
Formaldehyde (37%)
6.0 J.ll
12.5 J.ll
2.5 J.ll
4.0 J.ll