13. Prehybridization mix (add in order listed at 5 mL/100 cm2 membrane)
Conc. | 5 mL | 10 mL | 15 mL | x mL | Stocks |
5x | 500 礚 | 1000 礚 | 1500 礚 | 50x Denhardt誷 | |
5x | 1 mL | 2 mL | 3 mL | 20x SSC | |
50 mM | 250 礚 | 500 礚 | 750 礚 | 1 M NaPO4, pH 6.7 | |
50% | 2.5 mL | 5 mL | 7.5 mL | deionized formamide | |
250 礸/mL | 250 礚 | 500 礚 | 750 礚 | 5 mg/mL sheared salmon sperm DNA | |
1% | 100 礚 | 200 礚 | 300 礚 | 50% dextran sulfate MW 5000 | |
350 礚 | 700 礚 | 1050 礚 | Type I water | ||
0.1% | 50 礚 | 100 礚 | 150 礚 | 10% SDS | |
5 mL | 10 mL | 15 mL | x mL |
5x Denhardt誷, 5x SSC, 50 mM NaPO4, pH 6.7, 50% deionized formamide250 礸/mL sheared salmon sperm DNA, 1% dextran sulfate MW 5000 and 0.1% SDS
Prep the hyb mix and prehyb mix fresh each time. The hyb mix can sit at room temperature during the 3 hour prehyb step.
14. Hybridization mix (add in order listed at 5 mL/100 cm2 membrane)
Conc. | 5 mL | 10 mL | 15 mL | x mL | Stocks |
1x | 100 礚 | 200 礚 | 300 礚 | 50x Denhardt誷 | |
5x | 1 mL | 2 mL | 3 mL | 20x SSC | |
20 mM | 100 礚 | 200 礚 | 300 礚 | 1 M NaPO4, pH 6.7 | |
50% | 2.5 mL | 5 mL | 7.5 mL | deionized formamide | |
100 礸/mL | 100 礚 | 200 礚 | 300 礚 | 5 mg/mL sheared salmon sperm DNA | |
5% | 500 礚 | 1000 礚 | 1500 礚 | 50% dextran sulfate MW 5000 | |
650 礚 | 1300 礚 | 1950 礚 | Type I water | ||
0.1% | 50 礚 | 100 礚 | 150 礚 | 10% SDS | |
5 mL | 10 mL | 15 mL | x mL |
14. Hybridization mix (add in order listed at 5 mL/100 cm2 membrane):
1x Denhardt誷, 5x SSC, 20 mM NaPO4, pH 6.7, 50% deionized formamide, 100 礸/mL sheared salmon sperm DNA, 5% dextran sulfate MW 5000, and 0.1% SDS
15. Wash square Pyrex dish that will fit in shaking waterbath. Soak in 3% hydrogen peroxide or 0.1% DEPC for 10 min.
16. High Stringency Washes 1, 2 and 3
Wash 1 2x SSC | Wash 2 1x SSC | Wash 3
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