|dNTP (including biotin-11-dUTP|| |
|plasmid or genomic DNA|| |
|DNA polymerase I|| |
|Dnase I|| |
|Deionized or distilled water|| |
|Total reaction volume|| |
The most critical part of the labeling, if the DNA is clean, is the size of the final nick translation product. The best ISH results are obtained when the size of the biotin-labeled probe is around 300-600 bp. To determine the size of the probe, denature 15 µl of the nick translation product in boiling water for 4 min and cool down on ice immediately; and electrophorese the sample on a mini-gel with a DNA size marker. The size of the nick translation product can be adjusted by adding a different amount of DNase I in the reaction solution. Random primer labeling also can be used for biotin-labeling, however the size of the probe is relatively easier to control by nick translation.
The final volume of probe should be close to 55 µl. Usually, 5 µl of such a biotin-labeled probe (1 µg/55 µl = 18 ng/µl) is enough for one slide, and the probe can be used to process 100 slides. The size of the probe can be checked by electrophoresis on a mini-gel, either before or after the spin-colum purification. The probe can be stored in a freezer for several years.
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Kansas State University | Wheat Genetics Resource Center
31 January, 1997.