Bioinformatics |
Lab Protocol |
Malaysia University |
Malaysia Bank |
Email |
Plating Plaques
Contributor:
The Laboratory of George P. Smith at the University of Missouri
URL: G. P. Smith Lab Homepage
Overview
This protocol describes a standard method for plating and quantifying infectious units of wild-type phage as plaque-forming units. The plated phage can be used for library screening and titering, phage display experiments and a variety of other applications.
Procedure
1. Inoculate K91BluKan (see Hint #8) into 5 ml of NZY Medium in a 1 X 3 inch culture tube (see Hint #9).
2. Incubate on a rotary shaker at 27°C until the culture reaches mid-log phase, at which point the culture will appear turbid.
3. Melt TB soft agar and dispense 3 ml into two sterile glass tubes (13 x 100 mm) that have been prewarmed in a 50°C metal heating block (see Hint #10).
4.Pipette 10 to 200 μl of the appropriate phage dilutions (diluted into TBS/Gelatin) and a blank dilution as a control (see Protocol ID#2181) into 15 ml tubes.
5. Add 400 μl of the K91BluKan culture to each tube.
6. Pour each tube individually onto an NZY plate (see Hint #11) simultaneously with a soft-agar tubes from Step #3.
7. When the soft agar has hardened, incubate the plates at 37°C overnight.
8. Plaques should be visible the next morning for titering or library analysis.
Solutions
TBS/Gelatin
Store at room temperature
Autoclave 0.1 g Gelatin in 100 ml TBS
After autoclaving, swirl to mix in the melted gelatin 
TBS (1X)
50 mM Tris HCl, pH 7.5
Store at room temperature
Autoclave if desired
150 mM NaCl
TB soft agar
Add 100 ml ddH2O
1 g Bacto tryptone
Melt the agar in a microwave or a boiling water bath
0.75 g Bacto agar
Place the tubes in a 50°C temperature block to keep the agar molten.
Autoclave
Dispense 3 ml portions into sterile 13 x 100 mm tubes
To use for preparing plates:
Store at room temperature
0.5 g NaCl
Plates (1 liter)
While the agar is autoclaving, set out and label empty petri dishes
Dry overnight at room temperature or for a few hours in the 37°C incubator before use (see Hint #7).
Measure 500 ml of ddH2O into a 2 liter polypropylene erlenmeyer flask (see Hint #5)
Pour into plates (do not allow the temperature to fall below 50°C), adding at least enough to cover the bottom.
The medium is now at the average of room temperature and boiling (approximately 60°C). Antibiotics can be added at this temperature.
Add 11 g of Bacto agar
Cover with a polypropylene or glass beaker and autoclave.
Allow the plates to cool to room temperature.
Add 500 ml of 2X liquid Media (NZY or LB) (see Hint #6)
Also see Hint #4
NZY Medium (1X)
Adjust pH to 7.5 with NaOH
5 g NaCl
Autoclave and store at room temperature
Dissolve in 1 liter of ddH2O
Also see Hint #3
5 g Yeast Extract
10 g NZ Amine A
LB (1X)
Autoclave and store at room temperature
5 g/liter Yeast Extract
10 g/liter Tryptone
1 ml/liter 1.0 M NaOH
10 g/liter NaCl
Adjust pH to 7.0 with NaOH
Kanamycin Stock Solution
100 mg/ml Kanamycin Sulfate
Adjust pH between pH 6 to pH 8 with the appropriate base (NaOH) or acid (HCl)
Prepare in ddH2O
Filter sterilize and store at 4°C
IPTG Stock Solution
0.2 M IPTG (isopropylthio-β-D-galactoside)
Prepare in ddH2O
Filter sterilize and store at 4°C
X-gal Stock Solution
Store at 4°C protected from the light in a dark glass bottle (see Hint #2)
Prepare in Dimethylformamide (CAUTION! See Hint #1)
2% (w/v) X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside)
BioReagents and Chemicals
Gelatin
Dimethylformamide (DMF)
NZ Amine A
IPTG
Hydrochloric Acid
Antibiotics
X-Gal
Yeast Extract
Bacto Tryptone
Bacto Agar
Sodium Chloride
Sodium Hydroxide
Kanamycin Sulfate
Protocol Hints
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. Dimethylformamide dissolves many plastics, including polystyrene.
3. The contributors of this protocol use this particular rich media routinely; however, other media can be used, such as LB. NZY has the advantage that NZ amine A (from Humko Sheffield Chemical, P.O. Box 630, Norwich, NY 13815) is much cheaper than tryptone.
4. This is the recipe for 1 liter or approximately 40 plates; scale up or down as appropriate. Two-fold concentrated (2X) liquid medium is made and autoclaved in advance, and stored at room temperature in polypropylene bottles (The contributors of this protocol find it easier to pour from polypropylene than glass while keeping the solution sterile because of the problem of "drip-back" when using glass).
5. Use a flask of at least twice the volume capacity of the final volume of the solution. Again, plastic is preferable to glass.
6. Pour the medium gently down the wall of the flask, which should be held at an angle to prevent the medium from splashing down directly into the agar. These manipulations are designed to minimize the formation of bubbles, which are exceedingly hard to remove. Mix the contents of the flask by gentle rotation, holding the flask at a shallow angle to promote mixing. Again, avoid bubbles.
7. Plates can be dried within a few hours by laying them upside-down, tilted on the edge of their lids in a sterile laminar flow hood.
8. The K91BluKan E. coli strain is excellent for propagating filamentous phage. When wild-type phage are used, the plaques are large. Small but visible plaques are seen with infective members of fd-tet family. The K91BluKan contain a stable Hfr, even without selection. The cells require only thiamine in minimal medium. The phenotype of the K91BluKan cells is Lac Z-, Kanamycin resistant, Lac Y-, LacIQ (super-repressor) lacZ Ω-donor.
9. Use a larger tube if plating more than 12 phage dilutions.
10. If the plaques are to be produced by phage containing the α fragment of the lacZ gene (e.g., M13 phage), add 60 μl of 2% X-gal and 25 μl of 0.2 M IPTG to the soft agar tubes within 5 min of plating. The plaques will appear blue.
11. If using phage containing the α fragment of the lacZ gene (e.g., M13 phage), LB plates are preferred.
