Southern hybridization generally adopts liquid-solid hybridization, that is, the probe is in liquid phase and the hybridized DNA is in solid phase. Hybridization occurs in a solution (hybridization solution) under certain conditions and requires a certain temperature. Hybridization bottles or bags can be used, and the liquid can flow continuously on the membrane. The mixture can be made by yourself or purchased from the company. Different hybrid liquid formulations vary greatly, and the hybridization temperature is also different. The following is the mixed solution formula:

PEG 6000 10%； SDS 0.5%； 6×SSC； 50% formamide The hybridization temperature of this hybrid solution is 42℃.

1. Pre-hybridization

Immerse NC membrane in 2×SSC for 5min, add hybridization solution (5ml for 8cm×8 cm membrane) into the hybridization bottle, and stick the back of the membrane on the wall of the hybridization bottle with the front facing the hybridization solution. Put it 5m5minto a hybridization furnace at 42℃, and raise the temperature of the hybridization system to 42℃ ... The DNA of salmon sperm (dissolved in water or TE for 5 minutes) was denatured at 65438 000℃ for 5 minutes, and quickly added into a hybridization bottle to make its concentration reach 65438 000μ g/ml. Continue hybridization for 4 hours. The function of salmon sperm DNA is to block the sites without DNA transfer on NC membrane, reduce the hybridization background and improve the hybridization specificity.

intersect

Pour out the pre-hybridized hybrid solution, replace it with an equal amount of new hybrid solution heated to 42℃, and add denatured salmon sperm DNA at the same time. Heat the probe at 100℃ for 5 minutes to denature it, and quickly add it to the hybridization bottle. Hybridization at 42℃ overnight.