Bio-Rad ReadySub-Cell GT Cells - Manual (Page 14)

2.5 Note on Blotting
Nucleic acids within the gel can be transferred to membranes using the techniques of Southern
and northern blotting. It is beyond the scope of this instruction manual to include blotting procedures.
Consult references #1 and #2 for blotting techniques. Bio-Rad offers a full line of nitrocellulose and positively
charged nylon membranes, as well as vacuum and electrophoretic blotting apparatus for Southern and
northern blotting.
Section 3
Gel and Electrophoresis Reagent Preparation
RNA agarose formaldehyde gels
For 100 ml of a 1% agarose formaldehyde gel prepare as follows:
62 ml of 1.6% melted agarose
20 ml 5x MOPS electrophoresis buffer (1x final concentration)
18 ml 12.3 M (37.5%) formaldehyde (2.2 M final concentration)
Caution: Formaldehyde solutions and formaldehyde vapors are toxic. When handling solutions or gels
that contain formaldehyde use a chemical hood. Always wear gloves, safety glasses, and a laboratory
coat when using formaldehyde. See the MSDS for safety information.
Nucleic acid electrophoresis buffers 1—2
DNA agarose gel electrophoresis is usually performed using either Tris-Acetate-EDTA (TAE) or Tris-
Borate-EDTA (TBE). While TAE buffers provide faster electrophoretic migration of linear DNA and better
resolution of supercoiled DNA, TBE buffers have a stronger buffering capacity for longer or higher
voltage electrophoresis runs. Bio-Rad offers premixed 50x TAE and 10x TBE buffers for use with the
Sub-Cell GT systems. RNA formaldehyde gels require a MOPS [3-(N-morpholino)-propanesulfonic acid]
electrophoresis buffer.
1x Tris-Acetate-EDTA (TAE)–40 mM tris (pH 7.6), 20 mM acetic acid, and 1 mM EDTA.
50x Stock (1 liter)–dissolve in 600 ml distilled water:
242 g Tris base (FW = 121)
57.1 ml glacial acetic acid
100 ml 0.5 M EDTA (pH 8.0).
Fill to a final volume of 1 liter with distilled water.
1x Tris-Borate-EDTA (TBE)–89 mM tris (pH 7.6), 89 mM boric acid, 2 mM EDTA
10x Stock (1 liter)–dissolve in 600 ml distilled water:
108 g Tris base (FW = 121)
55 g boric acid (FW = 61.8)
40 ml 0.5 M EDTA (pH 8.0)
Fill to a final volume of 1 liter with distilled water.
1x MOPS Buffer (RNA Gels)–0.02 M MOPS [3-(N-morpholino)-propanesulfonic acid] (pH 7.0), 8 mM
sodium acetate, 1 mM EDTA (pH 8.0)
5x Stock (1 liter)–dissolve in 600 ml DEPC-treated distilled water:
20.6 g MOPS
13.3 ml 3 M sodium acetate (DEPC treated), pH 7.4
10 ml 0.5 M EDTA (DEPC-treated), pH 8.0
Fill to a final volume of 1 liter with DEPC-treated distilled water.
Caution: DEPC is a suspected carcinogen. Always wear gloves, safety glasses, and a laboratory coat.
Use caution when handling DEPC containing solutions. Consult the DEPC MSDS (Material Safety Data Sheet)
for more information.
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