Situ Hybridization by Boehringer

By Boehringer

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Sold under the tradename of Genius in the US. 47 4 Procedures for Labeling DNA, RNA, and Oligonucleotides with DIG, Biotin, or Fluorochromes ̈ 1 µl of either 1 mM DIG-dUTP (vial 3) or 1 mM Biotin-dUTP or 1 mM Fluorescein-dUTP. 5 (25°C)] (vial 4). 5 (25°C)] (vial 5). ̈ Enough redistilled H2O to make a final reaction volume of 20 µl. 4 Mix the reaction components well and © centrifuge briefly. 5 Incubate the tube at 37°C for 15 min. © 6 Place the tube on ice. 0) with 1 µl of glycogen solution (20 mg/ ml, in redistilled H2O) (vial 9).

Centrifuge the tube (at 13,000 x g) for 15 min at 4°C. ̈ Discard the supernatant. ̈ Wash the pellet with 100 µl ice-cold 70% (v/v) ethanol. ̈ Centrifuge the tube (at 13,000 x g) for 5 min at 4°C. ̈ Discard the supernatant. ̈ Dry the pellet under vacuum. Caution: Drying the pellet is important because small traces of residual ethanol will cause precipitation if the hybridization mixture contains dextran sulfate. Trace ethanol can also lead to serious background problems. 0) buffer. Note: If the hybridization buffer (used in Step 4 below) contains a high percentage formamide, dissolve the probe pellet in a smaller amount of TE buffer to form a more concentrated probe stock solution.

2 µl 1 mM DIG-dUTP or 1 mM Biotin-dUTP or 1 mM fluorochrome-labeled dUTP. ̈ 1 µg template DNA. ̈ 5 µl (5 ng) DNase I. ̈ 1 µl (10 units) DNA Polymerase I. Reagents available from Boehringer Mannheim for this procedure Reagent Description Cat. No. Pack size DIG-Nick Translation Mix* for in situ probes 5 x conc. 08 mM alkali-stable DIG-11-dUTP. 1 745 816 160 µl Biotin-Nick Translation Mix* for in situ probes 5 x conc. 08 mM biotin-16-dUTP. 1 745 824 160 µl Nick Translation Mix* for in situ probes 5 x conc.

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