Skim through the copy of the l ZAP cDNA library information sheeets attached to these instructions. As you will see, screening a phage library is a time consuming procedure that requires that some step in the procedure be done every day for about two weeks. Obviously, it is not practical for us to do this but it is worthwhile for you to have the experience of plating phage, lifting the placques off of the plates, hybridizing the filters and picking positive placques from the plates. Therefore you will plate a phage library that has been "spiked¹" with a pre-selected positive clone. Since we do not have time to do multiple platings we will plate the "spiked" library at a density that will allow you to isolate the clone. You will lift placques onto nitrocellulose filters, UV cross-link the DNA to the filter, pre-hybridize and hybridize the filter. You will then identify and pick a positive placque, and perform the in vivo excision of the phagemid. Subsequently you will subclone the cDNA into an expression vector.

Phage incorporating a genomic or cDNA library must be plated with special host bacteria that the phage are competent to infect. The host bacterium that we will be using is the XL1-Blue strain of E. coli. An overnight culture of XL1-Blue cells will be available. Someone from each laboratory group must start a liquid culture of these cells 4 - 4.5 hours before the class begins.

After 3-4 hours check the OD₆₀₀ of the cells. Allow the cells to grow to an OD₆₀₀ of 0.5 - 0.6. Pellet the bacteria at 3000 RPM for 10 minutes. Gently re-suspend the cells in a volume of 10mM MgSO₄ that is equal to the volume of the liquid culture (3 ml minus whatever you used to measure OD₆₀₀). Store the cells on ice until you are ready to use them.

After the bacteria are infected with phage they are plated in a low concentration agarose solution rather than on the top of a culture plate. The agarose must be kept melted but not so hot that it kills the bacteria. The ideal temperature is 48˚C. At temperatures below 45˚C the agarose will solidify; temperatures above 48˚C will kill the bacteria. You will be given a bottle of 0.7% NZY agarose. It must be melted in the microwave. Break up the solid mass of agarose with a sterile instrument before you place it in the microwave. Heat on high for about 1 minute.

After the agarose liquefies, aliquot 3 ml into each of 5 separate sterile culture tubes and place the tubes in a water bath equilibrated at 48˚C. You will need one tube for every plate you will be pouring plus one for practice (5 plates - see below).

When last tested the titre of the phage library that you will be using was such that a 1:10,000 dilution of the phage would give "perfect" plates for our purposes. I will have on hand a series of plates that show several different dilutions. Since we will be pipeting small volumes that are prone to error, we will plate several different dilutions of the phage. A small vial of full strength phage will be available. Use 100 µl of this to prepare a 1:10 dilution in SM buffer. From this original dilution prepare a series of dilutions from 10^-2 to 10^-6. You will plate each of these dilutions.

Mark 5 sterile culture tubes 10^-2 to 10^-6. Place 200 µl of the re-suspended cells in each of these tubes. Add 10µl of the appropriately diluted phage to the marked tubes. Incubate at 37˚C for 15-20 minutes.

While your bacteria + phage are incubating, mark 5 NZY plates 10^-2 to 10^-6. Use one aliquot of melted agarose to practice the pouring technique that will be demonstrated. After the incubation add the melted agarose to the infected bacteria then quickly vortex and pour the mixture as a smooth layer onto the plates. This must be done quickly or the agarose will solidify. Incubate the plates overnight at 37˚C. Someone from your group must take the plates out of the 37˚ incubator on the following morning and place them in the refrigerator.

Choose a plate in which all (or almost all) of the placques are well separated and can be picked individually. Pick the plate with the greatest number of separated placques.

Your plates will have been in the refrigerator for several days. In "real life" (if the life of a scientist can ever be called that) it is preferable to lift placques on the day after plating. It is important to place the plates in the refrigerator for about 2 hours to allow the top agarose to solidify well. Mark two nitrocellulose filters with your initials plus the dilution of the plate you choose for lifting. At each bench someone should prepare 3 wash trays containing denaturing solution (1.5M NaCl, 0.5 M NaOH), neutralization solution (1.5 M NaCl, 0.5 M Tris-HCl) and 2X SSC in separate trays. Use forceps and wear gloves to handle the filters. Place a filter on the plate; avoid wrinkles and bubbles. The filter should stay on the plate for two minutes. During this time place 3 non-symmetrical ink markers through the filter and into the agar plate. Use an ink-filled syringe and needle to do this. This is done so that you can eventually line up positive signals on your filter or film with placques on the plate. After two minutes gently lift the filter off the plate and place it in the denaturing solution (1.5 M NaCl, 0.5 M NaOH) for 5 minutes. Follow this with a 2 minute soak in the neutralization solution (1.5 M NaCl, 0.5 M Tris-HCl, pH 8.0) and a 30 second rinse in the 2X SSC. Place the filter DNA side up on blotting paper. As soon as you finish lifting the first filter, place the second filter on the same plate, mark this filter with ink, using the position of the original marks that are now on the plate for this filter also. This time leave the filter on the plate for 5 minutes. The rest of the steps are the same. UV crosslink the DNA to the filters. Do not allow the filters to dry completely before you crosslink them. Store the filters in a plastic bag between two pieces of blotting paper. Mark your bag. Seal the plates from which you lifted phage with parafilm and store it in the refrigerator.

see separate protocol

After you have hybridized your filters with the labeled probe (radioactive and non-radioactive) and carried out the detection procedure (film or reaction with strepavidinalkaline phosphatase and NBT/BCIP), you must align your filters with the plates and pick the positive plaques. For the radioactively-labeled filters, you must first line the film up with the filters and mark the placement of the ink dots on the film. This will be done for you since the filters are still radioactive. The marks on the film corresponds to the marks on the filter when it is facing "DNA-side" up. Turn the film over so that it is oriented with "DNA-side" down. Align the ink dots on the plates with the ink marks on the film, locate the positive plaques. Place the non-radioactively labeled filter on the bench with the DNA-side facing up. Place the phage plate up-side down over the filter and align the ink dots. Identify the plaques that gave a positive signal. Mark the positive plaques by circling them with marker on the underside of the plate. The same plaques should be positive for the two filters. Once you have identified the positive plaques, isolate a plaque along with the underlying agar using the blunt-end of a glass pipette. Transfer the plaque to a test tube containing 1 ml of SM buffer and 20µl of chloroform. Why is chloroform added to the SM buffer? The plaque must elute for 2-4 hr.

in vivo Excision of pBluescript from l ZAP

Stratagene's l ZAP vector has been designed to allow the insert cloned into the l vector to be excised in vivo and recircularized to form a phagemid containing the cloned insert. This in vivo excision is dependent on DNA sequences that were placed in the phage, including all of the sequence of the phagemid, pBluescript KS(-), and on the presence of a number of f1 bacteriophage-derived proteins. The proteins from the f1 phage recognize a region of the DNA that normally serves as the f1 bacteriophage origin of replication for positive strand synthesis. The site of inition and the site of termination for DNA synthesis have been subcloned separately into the l ZAP vector. The l ZAP clone is made accessible to the f1 derived proteins by simultaneously infecting bacteria with the l ZAP vector and f1 bacteriophage. Inside the bacteria the f1-dervied proteins recognize the site of initiation in the l ZAP vector. These proteins nick one of the two DNA strands. At the site of this nick, new DNA synthesis begins and continues through the sequence of the phagemid, pBluescript SK(-) and through the cloned insert to the termination signal which is placed 3' to the insert. The single-stranded DNA is circularized by an f1-derived protein, forming a circular DNA which contains pBluescript SK(-) and the inserted DNA and none of the phage DNA. Sequences within the f1 termination site permit the circularized DNA to be "packaged" as a filamentous phage particle and secreted from the bacteria. Once the phagemid is secreted, the bacteria are destroyed by heating, and the phagemid is allowed to infect fresh bacteria. These are spread LB/amp plates. Colonies represent clones of bacteria that have been transformed with Bluescript KS plus the inserted DNA.

in vivo Excision Protocol

_____________________________________________________________________________

Add deionized H₂O to final volume of 1 liter

Add deionized H₂O to a final volume of 1 liter
Adjust the pH to 7.5
Autoclave