Easy Way to Clone Genes From a Phage Library

互联网2013-09-06

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Easy Way to Clone

The protocol is oriented towards a C. albicans genomic library I made in Lambda Zap II on 7/97.

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.

Plaque lift unit

• Put phage plates to 4 degrees to firm up the top agar.
• Prepare the solutions; NaOH, Tris, and SSC, and pour them out on Whatman paper (two layers) for wetting the filters. You want the paper to be very wet, but not with free fluid swimming on top. When putting filters on, you don't want significant air bubbles beneath them.
• Write an identifier with pencil on each filter. Consider making duplicate filters for each plate to pre-verify that any signal in the end is real.
• Carefully lay the filter face-down over the plaques, then stab the sandwich with a high-guage syringe soaked in india ink in four places down to the plate bottom. This will provide good alignment marks for pulling plaques in the end.
• Carefully pull the filter back off the plate, and lay face-up on the NaOH solution paper for 5 minutes (all these times are rough). Then transfer to the Tris neutralization paper for 5 minutes, repeat on fresh Tris neutralization paper, then to 2X SSC for 5 minutes. You may repeat on 2X SSC.
• UV-crosslink in a Stratalinker while the filter is moist, then submerge in 2X SSC (or more dilute) to remove debris from the filter surface.
• Store dry for the long-term, or wet for immediate use.

Hybridization unit

• For fresh filters, prehybe in Church plus BSA for 30 minutes or more, warm. Old filters do not have to be prehybe'd, but may need to be boiled in the presence of the new probe to remove the old probe.
• Seal in a rotor unit, or if you have a lot of filters, you need to use a seal-a-meal bag, with about 1.5 to 2 ml hybe solution per small plate-sized filter. (I use 20 ml for 12 filters)
• Put to 65 degrees for 3 hours to overnight or longer, with sufficient agitation to expose all the filters.
• Save the hybridization solution on a 50 ml Falcon tube for future use. Wash the filters three times five minutes with church wash at room temperature in a separate container. The first wash is disposed in radioactive waste, subsequent ones go down the sink (with rinsing).
• Lay out on a film back and expose to X-Omat AR film for time as indicated by the geiger counter.
• Make sure to attach phosphorescent or other alignment marks to your assembly to allow precise alignment in the end.

Plaque pulling unit

• Congratuations! You got signal. Now you need to pull the plaque or plaque area to recover and purify the phage. Go to the light box with the autoradiogram, the filter assembly, and a marking pen. Align the filter assembly over the film and mark positive signals on the top saran wrap. Now get out the original plates from the refrigerator and go to your desk. With a right angle protractor, align the plate
with the filter assembly by their ink marks, then mark the signal location on the back of the plate with a pen.
• Prepare eppendorf tubes with .8 ml of SM for each postive signal you wish to pull.
• Take a long pasteur pipette and sqeezing gently on the bulb, jab into marked spot on the plate. Now withdraw gently, releasing the bulb to apply a bit of suction. Blow the plug into its destined SM tube. Mix thoroughly/let sit for a while to release the phage. This solution will be stable in the refrigerator for years, especially with a drop of fresh chloroform added to kill the bacteria.
• Now to purify the phage, some of the plug supernatant solution needs to be diluted and plated out to provide nicely separated plaques that can be re-probed (with the same hybe solution). Dilute 10 microliters supernatant to 1 ml of SM, and then plate out 1, 5 and 25 microliters from this dilution. It is bad form to let phage grow for too long (it leads to mutagenesis). For long-term storage of phage, combine some phage super with 20 percent DMSO, and freeze. Now go back to the other protocols.

Plasmid excision unit

• The biggest virtue of this Lambda Zap II phage library is that its inserts can be easily excised into plasmid (Bluescript) form. This procedure appears much more complicated than it actually is. First you must have the SolR cells grown up and prepared for phage plating, and get out some Exassist phage as well from the freezer.
• In a 15 ml falcon tube, combine 200 microliters XL blue cells and 200 microliters of phage supernatant and 1 microliter Exassist helper phage. Put to 37 degrees for 15 min.
• Add 3 ml LB and put to 37 degrees for 3 hours, shaking (1 hour is less efficient, but OK).
• Place to 65-70 degrees for 20 minutes to kill off the bacteria, then spin 15 min in the RC3 centrifuge at top speed.
• Add 100, 10 and 1 microliters supernatant to 200 microliters SolR cells in 1.5 ml tube. Put to 37 degrees for 15 minutes.
• Plate on LB amp plates.

SOLUTIONS

• Top agar: Tb-top is a simple recipe: 5g Tryptone, 2.5g NaCl, and 4g agar in 500 ml- autoclave, and add MgSO4 to 10mM and pour into 100 ml bottles for future microwaving. (Top agar and other large-scale phage growth solutions do not have maltose because it is not needed when infecting cells with a vast local excess of phage, and you don't want to lose phage in the end to binding to the cell debris.)
• SM: This is a stabilization and storage solution for phage. 2.9g NaCl(50 mM), 1g Mg SO4(10 mM), 25 ml Tris pH 7.5 (50 mM), 50 mg gelatin (100 g/l) in 500 ml-- autoclave.
• NaOH for plaque lifts: 0.5M NaOH, 1.5M Na Cl, 1 mM EDTA.
• Tris for plaque lifts: 0.5M Tris HCl pH 8, 1.5 M NaCl, 1 mM EDTA.
• SSC for plaque lifts: 2X, made from 20X lab stock- See Maniatis, etc.
• Church hybridization solution: (PNAS 81:1992) 1 percent BSA, 1mM EDTA, 7 percent SDS, and
0.5 M NaPO4 (From a 1.0 molar stock, pH 7.2: 134g Na2HPO4/7H2O plus 4.5 ml of 85 percent phosphoric acid to one liter; pH carefully, or else your hybe solution will be viscous, and that is wrong).
• Church wash: 50 to 100 mM NaPO4, 2 percent SDS, 1 mM EDTA.