, Pour ~ 350 mL of the low stringency wash solution into a Pyrex glass dish
, Write on a 50-mL Falcon centrifuge tube "Name of the hybridization solution
, Carefully, pour the hybridization solution into the labelled 50-mL Falcon centrifuge tube
, Store the hybridization solution in a 50 mL Falcon centrifuge tube in a-20 o C freezer
, Remove the blots from the bag. Immediately, place the blots one-by-one into a Pyrex glass dish containing 350 mL of low stringency wash solution
, Place the Pyrex dish on an orbital shaker and turn the speed dial to ~ 50-75 rpm. Wash the blots at room temp for 15 minutes
, After 15 minutes of washing, use a blunt-end forceps to hold the blots and pour the washing solution into a liquid waste radioactive container behind a plexi-glass shield
, While washing the blots in the low stringency wash solution, prepare another Pyrex dish containing ~ 350 mL of high stringency wash solution. Leave this dish in a 60 o C water bath incubator
, After 15 minutes of the second low stringency wash, transfer the blots into the Pyrex dish containing 350 mL of high stringency wash solution at 60 o C using blunt-end forceps. Wash blots in the high stringency wash at 60 o C for 30 minutes
, Use the Geiger counter to monitor the amount of labeled probes that remained on the dish. Wash the dish with a 7x Detergent solution and rinse it with water until radioactive lable, While washing the blots, pour off the low stringency wash solution into the liquid waste radioactive container
, Repeat the high stringency wash with a fresh 350 mL of high stringency wash solution
, While washing, cut out a piece of 3MM Whatman paper to a size of 8 inch x 10 inch. Wrap the paper with a piece of plastic wrap
, After washing the blots, briefly blot the blots on two or three layers of Kimwipe tissues to remove excess liquid
, In the lab, place the wrapped blots facing an intensifying screen in a vinyl X-ray cassette
, Bring the cassette, 2 pieces of small plexi-glass plates, 4 clamps, and a box of X-ray film to the darkroom on the first floor
, In the darkroom, remove a piece of X-ray film from the box
, Bend an upper right corner of the film to mark the orientation of the film relative to the blots
, Put X-ray film between the blots and the intensifying screen in the cassette
, Close the cassette
, Expose the blot to X-Ray film in a-80 o C freezer from several hours to a few days depending on how hot the blot is
, Developing the Exposed X-ray Film (Autoradiography)
, Remove the cassette from the-80 o C freezer and leave it in the lab until the cassette warms up to room temperature (~ 15-30 minutes)
, Take the cassette to the darkroom
, Develop the exposed X-ray film (autoradiogram) using the Kodak Film Developer
, Align the autoradiogram to the blots. Mark well positions on the blots to the autoradiogram. Write on the autoradiogram positions of the 1-kB ladder
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