Quantitative Nonisotopic Immuno-Dot-Blot Method for the Assessment of Cellular Poly(ADP-ribosyl)ation Capacity
✍ Scribed by Ragen Pfeiffer; Christine Brabeck; Alexander Bürkle
- Publisher
- Elsevier Science
- Year
- 1999
- Tongue
- English
- Weight
- 95 KB
- Volume
- 275
- Category
- Article
- ISSN
- 0003-2697
No coin nor oath required. For personal study only.
✦ Synopsis
did not appear to migrate out of the gel, causing band aggregation toward the bottom.
We also used the Spreadex gels in conventional gel tanks such as the BRL Horizon 11.14. without buffer recirculation or temperature control; results were much inferior to those obtained with the instrument used here, the Elchrom SEA 2000 apparatus. Electrophoresis at high temperature improved resolution and band sharpness compared with runs at ambient temperatures. The amount of PCR product needed for detection was comparable to or less than that required using silver staining of polyacrylamide gels, with syber green being superior to ethidium bromide and giving clearer background. The narrow resolving range of each gel type maximized fragment separation. While the 26-well format gave sharper bands, the 50-well format was optimal for analysis of high sample numbers. In both cases we were also able to perform simultaneous analysis of 2 loci where PCR products were of differing size but still within the range of one gel type.
We are currently using these gels in a study involving the microsatellite analysis of over 100 breast cancer patients at over 20 individual loci. Assuming no repeats, this could be achieved with 40 -80 Spreadex gels in comparison with about 70 -130 sequencing gels (depending on whether 2 PCR products are run together) and almost 200 polyacrylamide gels of the type we have described previously (1). Added to this ease and simplicity of usage, Spreadex gels present a significant advance in electrophoretic analysis of DNA.