Experimental preparation of λ phage particles from large-scale lysates
λ phage prepared from mass cultures can be recovered from the lysate by polyethylene glycolysis (PEG) precipitation in the presence of high salt. The residual cellular debris and PEG can be extracted with chloroform. Before further purification, it is recommended to determine the yield of the phage preparation. The source of this experiment is "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Experimental preparation of λ phage particles from large-scale lysates
Principle
λ phage prepared from mass cultures can be recovered from the lysate by polyethylene glycolysis (PEG) precipitation in the presence of high salt. The residual cellular debris and PEG can be extracted with chloroform. Prior to further purification, it is recommended that the yield of the phage preparation be determined.
Materials and Instruments
E. coli culture Move I. Materials For more product details, please visit Aladdin Scientific website.
Chloroform NaCl Polyethylene glycol SM Tryptic DNase I
Sorvall GSA rotor or equivalent Measuring cylinder
1. Buffers and solutions
Chloroform, NaCl ( solid), polyethylene glycol (PEG 8000) (approximately 50 g per 500 ml culture), SM.
2. enzymes and buffers
Tryptic DNase I ( 1 mg/ml), Tryptic RNase ( 1 mg/ml) in TE (pH 7.6).
3. centrifuge and rotor
Sorvall GSA rotor or equivalent.
4. Specialized equipment
Measuring cylinder (2 L).
5. Carriers and strains
E. coli culture, infected and lysed by λ phage.
II Methods
Precipitation of phage particles with PEG
1. Cool the lysed culture containing λ phage to room temperature, add tryptic DNase Ⅰ and RNase to a final concentration of 1 μg/ml, and incubate at room temperature for 30 min.
2. Add 29.2 g of solid NaCl (final concentration 1 mol/L) to every 500 ml of culture and stir to dissolve. The cultures were incubated in an ice bath for 1 h.
In the re-precipitation of phage particles with PEG, the addition of NaCl promotes the separation of phage particles from cellular debris and is therefore required for efficient precipitation. Some researchers prefer to add NaCl at the same time as the PEG, in which case the centrifugation in step 3 can be omitted. It should be noted, however, that the simultaneous addition of PEG and NaCl is only effective in precipitating phage particles if the phage are well grown and their titer in the initial lysate culture is greater than 2X1010 pfu/mI.
3. Centrifuge the cultures at 11,000 g (8300 r/min in Sorvall GSA rotor) for 10 min at 4°C to remove cell debris, and mix the supernatants of the four cultures in a clean 2 L measuring cylinder.
4. Determine the total volume of supernatant collected and transfer to a 2 L flask, add solid PEG to a final concentration of 10% (m/V, e.g., 500 ml of supernatant with 50 g of PEG), and dissolve the PEG by stirring slowly with a magnetic stirrer at room temperature.
5. Transfer the phage/PEG solution to a polypropylene centrifuge tube, cool in an ice-water bath, and leave for at least 1 h to allow the phage pellet to precipitate.
6. Centrifuge at 11000 g (8300 r/min in Sorvall GSA rotor) for 10 min at 4°C to recover the precipitated phage, remove the supernatant, and leave the tube upside down and tilted for 5 min to allow the residual liquid to drain well. The residual liquid was removed by pipetting.
Extraction of bacterial fragments with chloroform
7. Gently resuspend the phage precipitate in SM using a wide-mouth pipette with a rubber bulb (8 ml of SM for every 500 ml of supernatant for Step 3), tilt the tube so that the SM completely covers and soaks the phage precipitate, and leave for 1 h at room temperature.
Thoroughly but gently rinse the entire wall of the centrifuge tube, as the phage precipitate will adhere to the wall, especially if the tube is old and dented. And if the phage is aspirated violently, it is easy to cause the breakage of its tail.
8. Extract the PEG and cellular debris from the phage suspension by adding an equal volume of chloroform and shaking gently for 30 s. Centrifuge at 4°C, 3000 g (4300 r/min in Sorvall GSA rotor) for 15 min to separate the organic and hydrophilic phases, and recover the hydrophilic phase containing phage particles.
To determine the yield, the phage suspension was analyzed by gel electrophoresis.