Analysis of nucleic acids

Blot hybridization Prehybridization and Hybridization: 1) Incubate the membrane in a hybridization bottle with 15ml Church for 1 hour at 65°C 2) Add 50µl of the denaturated labeled probe. 3) Incubate overnight at 65°C in the oven with moderate rolling.

Washing 1) Preheat washing Church buffer at 65°C before use. 2) Rinse quickly the membrane. 3) Incubate 3 times for 15 minutes the membrane in washing buffer at 65°C in the bottle 4) Do additional one washing in the washing buffer at 65°C in a tupperware (waterbath)†. 5) Seal the membrane in plastic wrap and place it in a phosphorimager cassette. Let stand at room temperature‡ for the appropriate time (e.g. overnight for a Southern Blot, 4 hours for a Northern blot).

Deshybridization. 1) Boil the blot 5 minutes in 2 liters of deionised water with 20 ml 10%SDS. 2) Wash the blot with deionised water

1 M NaxHyPO4 pH 7.2: - Na2HPO4 (1 M, pH 7.2) - NaH2PO4 (1 M, pH 7.2) Check pH that should be at 7.2

68ml 32ml

Church buffer (1 liter): 7% SDS, O.5M NaxHyPO4 pH 7,2, 1mM EDTA - SDS 70g of SDS - NaxHyPO4 (pH 7,2) 500ml 0.5M EDTA 200mL Filter sterilize Washing buffer (1 liter): 1% SDS, 50mM NaxHyPO4 pH 7,2 10% SDS 100mL NaxHyPO4 50ml

† ‡

This step is optional, all washings can be done in the bottle. If so, rinse the membrane before wrapping. Do not put phosphorimager cassette at -80°C not to destroy the screens.

Analysis of Nucleic Acids 6