John M. S. Bartlett.pdf - Bio-Nica.info

Digoxigenin 353
standard DNA is mixed with 1 mL of 1× TNE containing Hoechst dye No. 33258 and
added to the quartz cuvet. Concentration of the Hoechst dye is 0.1 or 1 µL/mL in 1X TNE,
depending on the concentration of the calibration standard.
5. Termination mixtures containing 7-deaza-dGTP are used to avoid band compression
artifacts when sequencing guanine–cytosine-rich regions.
6. Similar to PCR, the success of Taq cycle sequencing depends on buffer conditions,
especially the concentration of Mg 2+ ions and pH. Buffer conditions for PCR and cycle
sequencing may be optimized using commercial “optimizer” kits (e.g., K1220-01,
Invitrogen, Leek, Netherlands). We have used Taq polymerases from Perkin–Elmer and
Boehringer Mannheim with comparable success for both PCR and sequencing. Thermal
cyclers from Perkin–Elmer (TC 480) and Biometra (Trio Thermo block) also gave
comparable results in our hands. Sequencing protocols using smaller numbers of cycles,
e.g., 20 to 25 may reduce the intensity of shadow bands.
7. Do not extend blotting time and do not use higher weights.
8. Protect eyes and skin against ultraviolet light (wear goggles, mask, coat, and gloves)!
9. To save on reagents, hybridization bags should be tightly sealed. However, leave one “long
end,” because bags will need to be reopened and resealed during subsequent incubation
steps.
10. Blocking reagent (Boehringer Mannheim) should be prepared freshly before use. Dissolve
blocking reagent at 50°C in TBS, and let cool down to room temperature. Blots can be
stored in blocking solution at 4°C for a few days.
11. To avoid diffuse or spotty background, the volume of the substrate solution should be
sufficiently large to cover the nylon membrane completely. Also, avoid folds in the
hybridization bag. Sequencing bands should become visible within 15 min on incubation.
Do not move the membrane during that time. Because the visualization process involves an
enzymatic reaction, lowering the temperature of the incubation solutions may lengthen incubation
times. We recommend developing at room temperature and checking the intensity of
the stained sequencing bands by briefly lifting the dark cover from time to time.
12. The intensity of the sequencing bands may increase during the first few hours after the final
incubation has been finished (if alkaline phosphatase and NBT/X-phosphate are used for
visualization as in the present protocol). Therefore, the incubation with substrate solution
should be stopped as soon as the first sequencing bands are clearly readable. Important:
Stained, dried nylon membranes should not be exposed to sunlight because they will
bleach rapidly. However, stained membranes can be stored safely for many months in
the dark. For long-term documentation, we recommend taking photographs. Sequencing
results may also be documented permanently by photocopying stained blots onto paper
or overhead transparencies.
References
1. Kösel, S. and Graeber, M. B. (1993) Non-radioactive direct sequencing of PCR products
amplified from neuropathological specimens. Brain Pathol. 3, 421– 424.
2. Kösel, S. and Graeber, M. B. (1994) Use of neuropathological tissue for molecular
genetic studies: parameters affecting DNA extraction and polymerase chain reaction. Acta
Neuropathol. 88, 19–25.
3. Kösel, S., Egensperger, R., Mehraein, P., and Graeber, M. B. (1994) No association of
mutations at nucleotide 5460 of mitochondrial NADH dehydrogenase with Alzheimer’s
disease. Biochem. Biophys. Res. Commun. 203, 745–749.
4. Krishnan, B. R., Blakesley, R. W., and Berg, D. E. (1991) Linear amplification DNA
sequencing directly from single phage plaques and bacterial colonies. Nucleic Acids Res.
19, 1153.