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1. Prepare the above mixtures
2.Incubate for 2 hours @ 37˚C
3. Mix in 2 µl DNAse I
4. Incubate for 30 minutes at 37˚C
5. Add 0.8 µl 0.5 M EDTA pH 8.0
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8. Place @ -80˚C for 45 minutes to overnight
9. Centrifuge for 10 minutes at 12,000 rpm @ 4˚C
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11. Rinse with 200 µl prechilled 80% ethanol
12. Centrifuge for 5 minutes at 12,000 rpm@ 4˚C
13. Remove as much liquid as possible
14. Air dry for 5-10 minutes at room temp
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Anti-DNP-POD: Perkin-Elmer Cat#NEL747A001KTCat# NEL747A001KT. Comes with blocking powder.
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Fluorescein Labeling Mix: Roche cat# Cat# 11685619910.
20X NTP mix: 100mM NTP's obtained from Amersham Bioscience (cat# Cat# 27202501).
Mix: 10 µl each of ATP, GTP, CTP with 6.5 µl UTP in 13.5 µl nuclease free H2O
Resulting in: 20 mM each ATP, GTP, CTP, 13mM 13 mM UTP stock.
20X DNP-11-UTP stock: Obtain a 250 nmol / 25 µl stock of DNP-11-UTP from Perkin-Elmer (cat# Cat# NEL555001EA). Add 10.7 µl nuclease free H2O for a 7 mM stock
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1. Fix embryos overnight at 4˚C in 4% PFA/1X PF in1X PBS.
2. Wash twice in PBST (1X PBS, 0.25% tween-20).
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Mix well, and heat @ 68˚ C for one hour to get powder into solution.
Aliqout Aliquot and store at -20˚C, and once thawed, never refreeze.
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100 mL distilled H2O
Place ingredients in erlenmeyer a flask and set flask in a beaker of water. Stir on magnetic stirrer and then heat until dissolved.
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