Library screen-cDNA probe
SCREENING BACTERIOPHAGE ml PLAQUES BY HYBRIDIZATION
To screen a library of mammalian DNA (genome complexity, 3 x 109 bp), a total of at least 300,000 recombinant plaques must be examined (generally, we screen 1 x 106 plaques). Table 10.1 gives the maximum number of plaques that can be screened in culture dishes.
In the following example, the volumes given are suitable for screening approximately 50,000 plaques in a 150-mm-diameter petri dish.
1. Melt 0.7% top agarose in a microwave and equilibrate at 55°C. Prewarm 150 mm agar plates at 37°C.
2. Mix aliquots of a packaging mixture or bacteriophage ml stock containing up to 50,000 bacteriophage particles in a volume of 100 ml of SM or less with 0.3 ml of plating bacteria. Incubate at 37°C for 20 minutes in 15 ml conical tubes in a water bath.
3. Add 7.5 ml of molten (50°C) top agarose (0.7%) and pour onto a 150-mm agar plate. The plates must be dry, otherwise the layer of top agarose peels off with the filter. Usually, plates that have been dried for 30 minutes at R.T. in sterile hood work well.
Note: Be sure to use top agarose rather than top agar since the latter peels off even more easily than the former.
4. Incubate at 37°C until the plaques reach a diameter of approximately 1.5 mm and are just beginning to make contact with one another (8-12 hours). The plate should not show confluent lysis.
5. Optional: Chill the plates at 4°C for an hour to allow the top agarose to harden or keep at 4°C O/N.
6. Number dry, nonsterile nitrocellulose filters (S&N filters) with a special nitrocellulose filter pen.
7. At room temperature, place a dry nitrocellulose circle neatly onto the surface of the top agarose so that it comes into direct contact with the plaques. Be careful not to trap air bubbles. The filter should be handled with gloved hands; finger oils prevent wetting of the filter and affect transfer of DNA. Mark the filter in three or more asymmetric locations (one, two, and three stabs) by stabbing straight through it and into the agar beneath with an 18-gauge needle attached to a syringe containing waterproof black drawing ink.
Once in contact with the top agarose, the filter wets very rapidly and transfer of bacteriophage DNA occurs quickly. Therefore, do not move the filter once contact with the plate is made. The easiest way of placing the filter on the plate is to hold it by its edges, bending it slightly so that the middle of the filter makes contact with the center of the plate. Let wetting action pull the rest of the filter onto the plate.
Make certain that the keying marks are asymmetrically placed and that both the filter and the plate are marked. There must be enough ink on the plate to be easily visible when a second filter is in place. Large blotches of ink, however, are undesirable.
8. After 60 seconds, use blunt-ended forceps to peel off the first filter and immerse it, DNA side up, in a shallow tray of a denaturing solution (1.5 M NaCl, 0.5 M NaOH) for 60 seconds. Transfer the filter into neutralizing solution (1.5 M NaCl, 0.5 M Tris Cl [pH 8.0]) for 5 minutes. Rinse the filter in 2 x SSPE and place in on Whatman 3MM paper to dry.
9. Place a second (if necessary), dry filter onto the same plate and mark it with ink at the same locations. After 1-2 minutes, peel the filter off the plate. Denature the DNA and neutralize as described in step 7 above.
Generally, the first filter is left in contact with the plaques for 30‑60 seconds and subsequent filters are left on about 30 seconds longer or until the filter is completely wet.
If any top agarose peels off the plate with the filter, remove it by gently agitating the filter in the denaturing solution.
10. After all the filters are dry, wrap them between circles of Whatman 1MM paper. Fix the DNA to the filter by baking for 2 hours at 80°C in a vacuum oven. Overbaking can cause the filters to become brittle.
11. Hybridize the filters (O/N) to a 32P-labelled probe.
12. Wash and autorad as usual.
Note: Any filters not used immediately in hybridization reactions should be wrapped loosely in saran wrap or sealed in hybridization quick seal bag.
Table 10.1 NUMBERS OF PLAQUES IN CULTURE DISHES OF VARIOUS SIZES
Volume of indicator Volume of Maximum
Total area bottom agar bacteria top agarose number of
Size of dish (cm2) (ml) (ml) (ml) plaques/dish
90-mm petri dish 63.9 30 0.1 3 15,000
150-mm petri dish 176.7 80 0.3 7.5 50,000
Benton and Davis (1977)
SM solution: 5.8 g NaCl
0.98 g MgSO4 (anhydrous) Note: Can add 0.01% gelatin
50 ml 1M Tris, pH 7.5
dd H2O to 1 L