Biomedical Engineering Reference
In-Depth Information
Important!
Do not touch the membranes with unprotected fingers,
use tweezers and gloves!
Squeeze out air bubbles using a glass rod. Moisten the membrane
with Soln. D and place five sheets of filter paper of gel size onto
the membrane. Cut paper towels to the same size as the membrane
and stack these on top of the filter paper to a height of about 4 cm.
Lay a glass plate on top of the pyramid and place a weight on top
to hold everything in place. Transfer at RT overnight.
Remove paper. Mark the position of the gel and the contact side
between gel and membrane on the membrane with a pencil. Rinse
the membrane with 1:10 diluted Soln. D and allow the membrane
to dry on air. Cover the membrane on both sides with filter paper,
fix filter paper and membrane with paper clips, and bake for 2 - 3 h
at 80
◦
C.
The baked nictrocellulose is ready for hybridization experi-
ments.
If other membranes are used instead of nitrocellulose, read their
instructions for use.
References
Southern EM (1975) J Mol Biol 98:503
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory
manual. 2nd. ed., vol. 2, Cold Spring Harbor Laboratory Press
Brown T (1999) In: Current protocols in molecular biology. Wiley, New
York, Unit 2.9.1
2.6 Drying of Electrophoresis Gels
The best way for storage of polyacrylamide gels is drying in vacuo.
Photograph or scan the gel before drying.
A lot of gel dryers are commercially available which allow to
perform the drying process at elevated temperature resulting in
shortened time.
Wetasheetoffilterpaperalittlebitlargerthanthegelwith
5% glycerol in water. Place this sheet on the porous surface of the
apparatus, lay the gel onto the paper, and cover it with wetted
cellophane or another type of plastic foil.
Important!
Coveronlyonesideofthegelwithawater-tightfoil!
Cover the gel with the rubber cloth, connect to the vacuum pump,
and switch on the heating. When the temperature at the position
of the gel is the same as that of the surrounding temperature,
continue drying for about 30 min and then open the apparatus. If
the vacuum is switched off too early, the gel becomes destroyed
irreversibly. To prevent this cracking, especially of gels with high
