Multi-Nuclease Protection Assay Dominik Haudenschild February 25,1998
- GAPDH 44 -> 36 Mouse/Rat/Human/Bovine
- Collagen II 33 -> 29/30/25.. Mouse/Rat/Human/Bovine/Horse
- Aggrecan 28 -> 26./27. Mouse/Human/Rat/Dog
Kinase-Max™ Oligo Labeling
- __5_µl Water (Nuclease-free)
- __1_µl 10pmol Oligo (at 10 pmol/µl = 10µM)
- __1_µl [g32P] ATP at 150 mCi/ml (NEG-035C)
- __2_µl 5x forward buffer
- __1_µl T4 PolyNucleotideKinase (10U/ml)
- Incubate at 37°C for 30 minutes
- Add 10µl gel loading buffer
- Boil 2 minutes at 95°C
- Load entire probe on 15% Acrylamide TBE/Urea gel
- Run at 200-250 volts for ~30 minutes
- Excise labeled probe
- Elute in 350µl of Probe Elution Buffer for 2hrs-overnight
- Measure 1µl in scintillation counter to determine activity
- Make probe cocktail that contains 100,000 DPM of each probe in 100µl
Multi-NPA Assay Dominik Haudenschild February 25,1998
- In a 1.5ml Hi-Yield microcentrifuge tube add:
- _____µl = 1 µg total RNA
- 100__µl = 100,000 counts of each probe
- _10__µl of 5M Ammonium Acetate, to 0.5M
- Also set up two tubes that contain:
- __1__µl of Yeast RNA (=5µg)
- 100__µl = 100,000 counts of each probe
- _10__µl of 5M Ammonium Acetate, to 0.5M