Patent ID: 6383756
Filing Date: 2002-05-07
Classification: C12Q

Abstract:
A method for detecting membrane-bound nucleic acids comprising:i) preincubating the membrane in a hybridization buffer consisting of 500 ml of 10Ã—TNT, 1250 ml of 20% SDS, 10 g of blocking reagent and 3250 ml of H2O; ii) hybridizing the nucleic acids with a non-radioactively labeled probe in said hybridization buffer; iii) washing at least twice in a buffer I consisting of 10 g of blocking reagent, 250 ml of 10Ã—PBS, 125 ml of 20% SDS and 4625 ml of H2O; iv) incubating the membrane with a streptavidin alkaline phosphatase conjugate in buffer I; v) washing the membrane once in buffer I, then washing at least twice in a buffer II consisting of 250 ml of 10Ã—PBS, 125 ml of 20% SDS, 4625 ml of H2O; then equilibrating the membrane in a buffer III consisting of 64.4 g of Tris HCl, 1770 ml of H2O, 210.4 g of diethanol amine, pH 9.75; vi) transferring the membrane to a solution of an alkaline phosphatase substrate in said buffer III; and vii) detecting a signal from a reaction of said alkaline phosphatase substrate with said alkaline phosphatase conjugate.