![]() * The probe concentration in the hybridization solution should be 10 ng/mL if the specific activity is 10 8 dpm/µg or 2 ng/mL if the specific activity is 10 9 dpm/µg. The bag must then be carefully be resealed to avoid leaking radioactivity. If using resealable bags the probe can be added by inserting a syringe into an uncut corner and injection of the probe. Pipet the desired volume * of probe into the hybridization tube and continue to incubate with rotation overnight at 42☌ (DNA-probe) or 60☌ (RNA-probe).Denatured probe should be added to the hybridization solution as soon as possible after denaturation. If the probe is dsDNA, denature by boiling for 5 minutes, and immediately place it on ice.If using resealable bags, it can be shaken or rocked slowly in a suitable incubator or water bath. Place the tube in a hybridization oven and incubate with rotation 15 minutes (3 hours if using a nitrocellulose membrane) at 42☌ (DNA-probe) or 60☌ (RNA-probe).Then a corner of the bag can be cut off to allow pipetting of the prehybridization/hybridization solution into the bag. In which case, the membrane should be sealed into a bag after being wetted. Alternatively one can use resealable bags. Place the membrane in a hybridization tube with the RNA-side facing in, and add ~1mL formamide prehybridization/hybridization solution per 10 cm 2. ![]() Wet the membrane carrying the immobilized RNA in 6X SSC.If the membrane was blotted from a glyoxal gel, soak the membrane in 20 mM Tris-HCl (pH 8.0) for 5 minutes to disassociate the glyoxal from the bound RNA.formamide prehybridization/hybridization solution.Membrane (nylon or nitrocellulose) with immobilized RNA.The hybridization step of a northern blot allows identification of RNA blotted and immobilized on a membrane with a radioactive (or otherwise labeled) nucleic acid probe. Adapted from Current Protocols in Molecular Biology ![]()
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