Adapted from Current Protocols in Molecular Biology and the Stratagene® instruction manual for HybriZAP Two-Hybrid cDNA Gigapack® Cloning Kit.
This stage of phage library screening includes plating large numbers of phage; this is done according to the library titer to yield high counts of individual plaques on several plates. The phage is then transferred to nylon membranes denatured and its DNA immobilized to the membrane. This allows subsequent hybridization with a labeled probe to indicate phage containing homologous DNA.
- Recombinant phage library dilution from titer.
- Host bacteria- XL1-Blue MRF' -freshly streaked onto an LB-Tet agar plate.
- 150mm (round 1) or 86mm (round 2+) NZY agar plates.
- 0.7% NZYM top agarose.
- NZYM broth.
- Suspension Media (SM)
- Magna-Lift® or other nylon transfer membranes for 150mm or 100mm plates.
- ~20-gauge needle and india ink
- Whatman 3MM paper
- 0.2 M NaOH/1.5 M NaCl
- 0.4 M Tris-HCl (pH 7.6)/2X SSC
- Pick a colony of XL1-Blue MRF' from the LB-Tet plate and grow to saturation in 5 mL of NZYM broth.
- If this is the first round of plating and lifting phage, it is necessary to determine the number of plaques that should be screened in order to come across your gene of interest. For our zebrafish cDNA libraries, a rare message can generally be found if you screen „ 300,000 plaques. The large (150mm plates) typically hold from 20,000-40,000 plaques. Generally, ten 150mm plates with a pfu=~30,000 will be sufficient.
- Melt NZYM top agarose in microwave and place in waterbath to equilibrate at 45-50°C.
- Label (numbering is sufficient) NZY plates and prewarm at 37°C.
- For each plate to be poured, combine the recombinant phage and plating bacteria (grown O/N in 1 above) in a sterile glass tube. Do not add the top agarose now. The amounts to be added are shown in the following table.
Mixtures for plating phage libraries
Saturated XL1-Blue MRF' in NZYM
0.7% NZYM top agarose
- Incubate at room temperature while gently shaking for 20 minutes to allow the bacteriophage to adsorp to the bacteria.
- Incubate at 37°C for 10 minutes. During this time the adsorped bacteriophages inject their DNA into the host bacteria.
- Add the NZYM top agarose (amount shown in above table) to each tube, briefly mix by vortexing, and pour onto NZY agar plates. Spread the agarose across the plate by tilting the plate gently.
- After all the top agarose/cells have been poured and set, move the plates to the 37°C incubator for 8-12 hours.Don't allow the plates to incubate too long. The plaques will grow quickly and superimpose.
- Remove the plates from the 37°C incubator and transfer to 4°C cold room for at least 1 hour to firm up plates prior to transfer.
- Prepare three separate sheets of Whatman 3MM on a piece of Saran-Wrap or in a shallow tray. Saturate one with 0.2 M NaOH/1.5 M NaCl, one with 0.4 M Tris-HCl (pH 7.6)/2X SSC, and one with 2X SSC.
- Label or number nylon membranes (one at a time works best) according to the system used for your plates with a ball point pen. Be sure to indicate which lift (if doing more than one per plate- up to 5 are acceptable) on each membrane as well.
- Place filter label side up onto plate. This is best done by touching the edge of the filter and progressively laying down more of the filter as it wets. Avoid bubbles.
- Leave filter on plate for 1-2 minutes (longer is OK). During the transfer record the orientation of the filter by dipping a ~20-gauge needle in India ink and stabbing it through the membrane into the agar at several asymetric exterior points.
- Remove the filter with a forceps and place on a dry sheet of Whatman 3MM paper to dry for 10 minutes.
- Transfer the filter to the Whatman 3MM paper saturated with 0.2 M NaOH/1.5 M NaCl and incubate 1-2 minutes.
- Transfer the filter to the Whatman 3MM paper saturated with 0.4 M Tris-HCl (pH 7.6)/2X SSC and incubate 1-2 minutes.
- Transfer the filter to the Whatman 3MM paper saturated with 2X SSC and incubate 1-2 minutes.
- Place filter on a dry sheet of Whatman 3MM paper and UV crosslink.
- Dry in a 37-42°C oven for ~20 minutes.
- Place filters between dry sheets of Whatman 3MM paper until needed for hybridization.
Long term storage should be at 4°C or under a vacuum