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Calcium Chloride Transfection of Bacterial Cells with Bacteriophage DNA
Contributor:
The Laboratory of George P. Smith at the University of Missouri
URL: G. P. Smith Lab Homepage
Overview
This protocol describes the introduction of bacteriophage DNA into bacteria. The procedure is generally used for phage display library construction and propagation of recombinant clones for DNA sequencing and other analyses. The host bacterial strain is female (F-; lacking pili) and therefore non-infectible. This procedure is similar to standard protocols on transformation of bacteria with Calcium Chloride (see Protocol ID#386) and includes steps that improve the yield of transformants carrying phage DNA.
Procedure
1. Inoculate a 1.7 ml NZY culture in a 13 ml plastic culture tube with a colony of fd-tet bacteriophage-infected bacteria. Incubate the tube vertically with shaking at 300 rpm overnight at 37°C.
2. Inoculate 20 ml of NZY in a 250 ml culture flask with up to 500 μl of the overnight culture. Grow the cells with vigorous shaking at 300 rpm at 37°C until the optical density (OD600) at 600 nm reaches 0.4 to 0.5 (see Hint #2).
3. Pour the culture into a sterile centrifuge tube and pellet the cells by centrifugation in a Sorvall™ SS-34 rotor for 10 min at 2,500 rpm (750 X g) at 4°C.
4. Decant the supernatant and gently resuspend the cells by trituration in 10 ml of ice-cold Calcium Chloride Solution. Incubate the tube on ice for 20 min.
5. Pellet the cells as in Step #3, and decant the supernatant. Gently resuspend the cells by trituration in 1 ml of ice-cold Calcium Chloride Solution. These cells are now "competent" to take up foreign DNA and may be stored at 4°C for up to five days.
6. For each transfection, add 200 μl of competent cells from Step #5, and add 200 μl of phage DNA into a sterile 13 ml plastic culture tube (see Hint #3).
7. Immerse the tubes halfway into a waterbath at 37°C for 30 sec.
8. Place the tubes on ice for 1 hr, swirling the tubes occasionally.
9. Immerse the tubes halfway into a waterbath at 42°C for 2 min.
10. Place the tubes on ice for 2 min.
11. Add 1.6 ml of pre-warmed (37°C) NZY Medium containing 0.2 μg/ml Tetracycline to the tubes on ice. Transfer the tubes to a shaking platform and incubate at 37°C for 30 to 60 min at 225 rpm (see Hint #4).
12. Perform serial dilutions of the samples using NZY Medium as the diluent (see Protocol ID#2181 for appropriate dilution range). Pipette up to 2 ml of each dilution into a separate 15 ml polypropylene tube.
13. Into each 15 ml tube pour 3 ml of molten TB Soft Agar and immediately pour the mixture (without vortexing) onto an NZY plate containing 40 μg/ml Tetracycline. After the soft agar has hardened, incubate the plates at 37°C for one to two days. Colonies will have a wide range of sizes depending on the depth in the soft-agar layer (see Hint #5).
Solutions
TB soft agar
Autoclave and store at room temperature
1 g Bacto Tryptone (Difco)
To use for pouring plates:
Into a number of glass 125 ml bottles weigh:
Add 100 ml of ddH2O
0.75 g Bacto Agar (Difco)
Dispense approximately 3 ml portions into sterile 13 X 100 mm plastic tubes and place the tubes at 50°C until ready to pour
Melt the agar in a microwave or boiling water bath
0.5 g NaCl 
Calcium Chloride Solution
Filter or autoclave to sterilize
100 mM CaCl2
Tetracycline (1000X)
40 ml of 40 mg/ml Tetracycline
Mix thoroughly and store at 20°C in a tube covered with aluminum foil
Filter Sterilize
Add 40 ml of autoclaved 100%(v/v) Glycerol
NZY Medium
5 g NaCl
For solid medium, add 20 g Bacto Agar (Difco) before autoclaving and pour into plates after autoclaving
Dissolve in 1 liter water
Store at room temperature
10 g NZ Amine A (Humko Sheffield Chemical)
Autoclave to sterilize
5 g Bacto Yeast Extract (Difco)
Adjust pH to 7.5 with NaOH (CAUTION! See Hint #1)
BioReagents and Chemicals
NZ Amine A
Tetracycline
Glycerol
Bacto Agar
Bacto Yeast Extract
Bacto Tryptone
Calcium Chloride
NaOH
Sodium Chloride
Protocol Hints
1. CAUTION! This substance is a biohazard. Please consult this agent's MSDS for proper handling instructions.
2. The time required will depend upon the amount of cells in the inoculum and other experimental conditions, and should be determined empirically.
3. Appropriate amounts of DNA are approximately: 5 X 106 molecules of untreated RF; 5 X 108 molecules of single-stranded viral DNA (ssDNA); 108 molecules of restricted vector ligated with target insert; 1010 molecules of double-stranded RF converted by T4 DNA polymerase and T4 DNA ligase from ssDNA hybridized with a mutagenic oligonucleotide (see Protocol ID#2172 for detailed explanations).
4. Cells that have been transfected with RF DNA will express the genes conferring resistance to Tetracycline during this incubation.
5. Much lower yields are achieved if the transfection mixture is spread directly on the plates without soft agar.
