Bio-Rad Sequi-Gen GT Sequencing Cell - Manual (Page 37)

7.3 Gel Drying and Autoradiography
The radiolabeled oligonucleotides may be visualized by a variety of techniques involving
autoradiography. For the best resolution and signal intensity, dry DNA sequencing gels
with a slab gel dryer.
1. Transfer sequencing gels to a fresh sheet of filter paper. Wet the gel slightly by misting the
gel with deionized H
2
O. Lay the dry filter paper on top of the gel, and press firmly. The gel
will stick to the paper. Pick up the gel by lifting the filter paper carefully from one end.
2. Cover the sequencing gel with plastic wrap. Smooth out air bubbles and folds by rubbing
with a paper towel, and trim the edges to fit the slab gel dryer.
3. Set Model 583 Gel Dryer to sequencing cycle. 30 minutes at 80 °C should suffice for
drying thin low percent gels, if the applied vacuum is above 28 inches of mercury or
125 torr. Refer to the dryer’s instruction manual for details.
4. Autoradiograph the gel with high speed X-ray film (such as Kodak XAR) and a suitable
film cassette. Intensifying screens are optional. If 35 S radiolabel is used, the gel can be
left on the outer glass plate and fixed in 1 liter of 10% acetic acid, 10% methanol for
15 minutes. This removes hygroscopic urea. The gel may then be dried on filter paper.
Removal of plastic wrap before autoradiography is important because
35 S is a weak beta
emitter. Autoradiography of 35 S labeled fragments typically requires 1–3 days. (However,
we have found the fixative step unnecessary, even when sequencing with 35 S.)
7.4 Nucleic Acid Separation Applications for the Sequi-Gen GT
Electrophoresis System
Several other nucleic acid separation techniques requiring single nucleotide resolution
can be conducted using the Sequi-Gen GT systems. Below is a comprehensive list. Refer to
Sambrook, J., Fritsch, E. F., and Maniatas, T., Molecular Cloning, A Laboratory Manual,
Second Edition, Cold Spring Harbor Laboratory Press, 1989, or Ausubel, F. M., et al., Current
Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Interscience, 1987,
for more information and protocols.
• Microsatellite Analysis
• Single-Strand Conformational Polymorphism (SSCP) studies
• Heteroduplex analysis
• DNA footprinting
• DNA fingerprinting
• RNase protection assays
• S1 nuclease mapping
• Primer extension studies
• DNA/Protein binding studies (gel shift assays)
• Oligonucleotide analysis
34