M.Fero — 11/94
- Prepare serial 10-fold dilutions of transformed bacteria in LB and spread 100 µL onto LB/Amp plates, as described in the Electroporation protocol. Incubate at 37°C overnight. Also streak out positive and negative control bacteria on a separate plate.
- Select plates that have an optimal density of bacteria (i.e. 2 mm spacing between colonies). Put selected plates at 4°C for one hour.
- Lay 83 mm circular nylon membrane on surface of agar plate until it becomes thoroughly wetted. Poke 3 widely spaced wholes in an asymmetrical pattern through the filters and the agar near the edge of the dish with a 18G or 21G needle for future orientation (see Figure 1).
- Place Saran wrap on benchtop. Spot out 0.5 mL puddles of denaturing solution (x1) and neutralizing solution (x2) for each filter at 6" intervals on Saran wrap.
- Using forceps, carefully peel nylon membrane from agar surface and place it colony side up onto a puddle of denaturing solution. The colonies should stick to the membrane and not to the plate. Incubate for 5 min. Blot it briefly on a paper towel (colony side up) and then transfer it to the first puddle of neutralizing solution for 5 minutes. Repeat this with a second puddle of neutralizing solution.
- Cross link the DNA under a UV light (Stratalinker).
- Rinse the filters in 2X SSC + 0.1% SDS on a rotator.
- Lay the filter colony side down onto a paper towel. Lay a second towel on top and gently press on the filter to blot away most of the lysed bacterial protein. Be careful not to smear the colonies. They can be kept moist or dry until hybridization.
- Regrow colonies on the plates by returning them to 37°C for 4 hrs and then store them at 4°C.
- Hybridize the filters and wash them as usual. Monitor the washes with a Geiger counter to be sure that most of the counts have been washed off. If an oligo is used as a probe then the hybridization should be done in the refrigerator without formamide and the washes should be done at low stringency (1x SSC) at room temperature.
- Dry the filters and lay them out on an old piece of film. Place fluorescent markers at the corners of the film. Wrap the film in Saran wrap to hold the filters in place. Expose to film at –70°C for 4 hrs to overnight.
- Develop and dry the autoradiograph. Line up the autorad with the filters such that the fluorescent markers line up precisely. Dot the locations of the filters pinholes on the autorad with a Sharpie. Place the autorad on a light box (see Figure 2). Place the bacterial dishes on the autoroad lining up the agar pinholes directly over the ink marks on the film. Pick colonies that correspond to the autorad spots.
LB Amp plates
Positive and negative control bacteria
Denaturing solution (0.5 M NaOH + 1 M NaCl)
Neutralizing solution (1 M Tris pH 7.4, 1 M NaCl)
2X SSC, 0.1% SDS
83 mm circular nylon membranes (Hybond N)
Stratalinker UV box
Hybridization Solution (e.g. Stark’s)
Autoradiograph film and cassettes