By DeMuro, Rapley
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Additional info for Gene Probes: Principles and Protocols (Methods in Molecular Biology Vol 179)
Example text
A positional match in the target string is indicated by = in the match window. 8. Change the Search depth to one or more mismatches, and click on MATCH. This time nontarget sequences with one or more mismatches to the target string will 16S rRNA-Targeted Probes for FISH 9. 10. 11. 12. 13. 33 appear in the match window below the sequences with an exact match to the target string. Positional mismatches are indicated by the nucleotide that does not form a canonical pairing with its target string complement.
Columns: For the removal of unincorporated nucleotides from the labeled DNA probe, we utilized the Quick Spin columns provided by Boehringer Mannheim (cat. no. 100408) following the manufacturer’s instructions. These are ready-touse disposable G-50 Sephadex columns that can otherwise be packed manually into syringe barrels, although this is a tedious and time consuming step. 8% agarose gels. Many vendors offer agarose for routine use (gelling temperature approx 36°C), and they were all satisfactory.
The percent incorporation can be calculated by dividing the counts from the washed filter by the counts from the unwashed filter and multiplying by 100. The percentage incorporation should be above 60%. 3 (3 µL of a 1:100 dilution) and the multiplying by 55 (the reaction volume). Specific activity is expressed as cpm/µg and a good reaction will yield around 10 8 cpm/µg. Although it is not essential to measure the percent incorporation and specific activity of probes before their use, it does enable the monitoring of reactions from one experiment to another.