Clone Genes From a Phage Library

The overall sequence of events is:

• Titer and plate out phage

• Lift plaques onto filters and prepare them for screening

• Make a probe

• Hybridize the probe to the filters

• Wash the filters and expose to film

• Purify putative plaques

• Excise plasmid from the desired phage

Some preparatory items

The library (one that I made from C. albicans genomic DNA) is kept at -80 degrees C in 0.5 ml aliquots. This solution is also stable in the refrigerator for several months at least. The library may be amplified if you wish, but this should not be necessary. It was made by cutting the genomic DNA with Tsp509 I at several different concentrations and size-selecting pieces from 4 to 8-9 kb from each digestion to ligate into the Lambda ZapII vector (at Eco RI). Its complexity is roughly 80,000 X an ｉｎｓｅｒｔ size of 4 to 8 kb = 320 Mbp, which is about 20-fold coverage of this yeast genome.

The aim is to evenly plate out about 10,000 phage over about 10 plates. What you need in addition to the phage is: Plates: LB, LB tet, LB Kan, LB amp; Top agar: NZY or TB plus 0.7 percent agar autoclave); Cells: XLBlue MR strain, which is tetracycline resistant (on an F prime element; 25 micrograms/ml tet), and the SolR strain, which is kanamycin resistant (use 25 micrograms per ml as well). Both of these strains should be recovered from the freezer onto the appropriate drug plate and then kept in the refrigerator. Grow the bacteria in LB + 0.2 percent maltose (to express the mal permease which is the lambda receptor) overnight at 30 degrees, spin down in a 50 ml Falcon tube, and resuspend to half volume in 10 mM MgSO4. These bacteria will be good to use for phage plating for several weeks.Use 50 microliters per plate. Filters: I like simple uncharged nylon filters from Fisher (MSI N00HY08250, 82 mm, 50/box). These are quite cheap. Solutions: LB, SM solution, Church hybridization solution, Church wash solution, Chloroform (see the end for recipes).Ex-assist phage as well for plasmid excision (from Stratagene).

Phage plating unit

• Put out the required LB plates to 37 or 30 degrees one to three hours before they are needed.

• When using a library for the first time, the first step is to titer the library, confirming the titer stated by whoever gave it to you. Dilute phage in SM solution to a series of test dilutions.

• Mix the phage desired (in about 1 to 50 microliters) with 50 microliters of bacteria and incubate at 30 degrees or 37 degrees for 20minutes.

• Place enough culture tubes at 45-48 degrees. This can either be in an electric block bath with appropriately sized holes or in an ad-hoc water bath of tap water in an ice bucket.

• Melt enough top agar in the microwave to aliquot three mls into each culture tube.

• Add the phage/cells to the top agar, vortex gently without frothing, and pour gently over the LB plate, tipping to cover it completely.

• Place at 37 degrees. Plaques will be apparent by 6 hours or so and ready to lift a few hours after that. For the original library plating, you want the plaques to be pretty small (1 to 2 mm), and you will probably use a high enough density so that the plaques will be confluent. For plaque purification, on the other hand, you will want to have relatively large (3 mm), well-separated plaques, which can be left to go overnight if you wish.