P1 Application of phages and their cloning systems
The P1 phage was discovered in the same year as the λ phage (Bertani 1951). Both phages are mild phages in their natural hosts, and their research history in the laboratory is almost the same. λ phage, once discovered, was immediately chosen by influential laboratories at that time as a model for studying lysogenic phages at the molecular level. The source of this experiment is "Laboratory Guide to Molecular Cloning, Third Edition" Translated by Huang Peitang et al.
Operation method
Application of P1 phage and its cloning system
Principle
The P1 phage was discovered in the same year as the λ phage (Bertani 1951). Both phages are mild phages in their natural hosts, and their laboratory history is almost identical. λ phage was immediately chosen as a model for studying lysogenic phages at the molecular level by the influential laboratories of the time once it was discovered.
Materials and Instruments
Restriction endonuclease E. coli strain Move I. Materials For more product details, please visit Aladdin Scientific website.
Ethanol IPTG Isopropyl Alcohol Phenol Chloroform Polyethylene Glycol Sodium Acetate DNA Isolates Alkaline Lysate TE
Pulsed-field gel LB medium TB medium Sorvall SS-34 Turning head or equivalent Corex tubes Water bath 65°C
1. Buffers and solutions
Ammonium acetate (0.5 mol/L)
Ethanol
IPTG ( 1 mmol/L)
Isopropyl alcohol
MgCl2 (1 mmol/L)
Phenol: chloroform (1:1, V/V)
Polyethylene glycol (40%, m/V, PEG8000 solution)
Sodium acetate (0.3 mol/L, pH 5.2)
DNA isolation solution
Alkaline lysis solution Ⅰ
Alkaline lysis solution Ⅱ
Alkaline cleavage solution Ⅲ
TE ( pH 8.0 )
TE containing 20 μg/ml RNase (pH 8.0)
2. Enzyme and buffer
Restriction endonuclease
3. gels
Pulsed field gel (or 0.5% agarose gel)
4. Culture medium
LB medium containing 25 μg/ml kanamycin.
TB medium containing 25 μg/ml kanamycin.
5. Centrifuges and rotors
Sorvall SS-34 head or equivalent.
6. Specialized equipment
Corex centrifuge tube (30 ml)
65°C water bath
7. Carriers and Strains
E. coli strains (cultures) transformed with non-recombinant P1 phage or PAC vectors
E. coli strains (cultures) transformed with recombinant P1 phage or PAC vectors.
II Methods
Preparation of recombinant P1 or PAC DNA
1. Add 10 ml of TB containing 25 μg/ml kanamycin into two 50 ml Falcon test tubes or Erlenmeyer flasks, inoculate a single colony containing recombinant P1 or PAC in one test tube, and inoculate a colony transformed with only the empty vector in the other test tube, and then shake vigorously at 37℃ for 12~16 h to saturate the culture.
2. Transfer the culture to a 15 ml centrifuge tube and centrifuge at 3500 g (Sorvall SS-34 turntable, 5400 r/min) for 5 min at 4℃, collect the cell precipitate, re-suspend the precipitate with 3 ml of sterile water, and repeat the centrifugation procedure.
3. Resuspend the precipitate in 2 ml of alkaline lysate I and place on ice.
4. add 3 ml Alkaline Lysate II, gently turn the tube over several times to mix the solution, and place on ice for 10 min.
5. Add 3 ml of ice pre-cooled Alkaline Lysate III to each tube of cell suspension, gently turn the tube several times, mix the solution, and place on ice for 10 min.
6. Centrifuge at 12000 g (Sorvall SS-34 turntable, 10000 r/min) for 10 min at 4°C to precipitate cellular debris.
7. Transfer the supernatant (~7 ml) to a 30 ml Corex tube, add an equal volume of isopropanol, and gently turn the tube several times to mix the solution. 12,000 g (Sorvall SS-34 turntable, 10,000 r/min) centrifugation at 4°C for 10 min is performed to collect the nucleic acid precipitate.
8. Carefully aspirate the supernatant and invert the centrifuge tube onto a Kimwipe tissue until no droplets escape. Remove the droplets from the walls of the tube with a Pasteur pipette attached to the vacuum pump and add 0.4 ml of 0.3 mol/L sodium acetate (pH 5.2) to dissolve the nucleic acid precipitate. heat briefly at 65 °C for a few minutes to aid in the dissolution of the nucleic acid.
Purification of Recombinant P1 or PAC DNA
9. Transfer the DNA solution to a microcentrifuge tube. Extract once with an equal volume of phenol: chloroform. Separate the aqueous phase from the organic phase by centrifugation in a microcentrifuge at maximum speed for 5 min at room temperature. After centrifugation, transfer the upper aqueous phase to a new microcentrifuge tube.
10. 1 ml of ice-cooled ethanol is added and the tube is turned several times to mix the solution. DNA is precipitated by centrifugation at maximum speed for 10 min at 4°C. The precipitate is washed with 0.5 ml of 70% ethanol and centrifuged again for 2 min at 4°C.
11. Carefully remove the supernatant, invert the centrifuge tube, and dry at room temperature until the traces of ethanol have evaporated. Add 0.4 ml of RNase-containing TE to the precipitate, cover the tube and place it at 37℃. Shake the tube gently and intermittently for the next 15 min to help dissolve the DNA. Continue to incubate for 2 h.
12. Add 4 μl of 1 mol/L MgCl2 and 200 μl of 40% PEG solution, mix well, and centrifuge at maximum speed for 15 min at 4°C to collect the DNA precipitate.
13. Aspirate off the supernatant. Resuspend the precipitate in 0.5 ml of 0.5 mol/L ammonium acetate, add 1 ml of ethanol, turn the centrifuge tube several times, mix well, and then centrifuge at maximum speed for 15 min at 4℃ to collect the precipitate.
14. Aspirate the supernatant again and wash the precipitate twice with 0.5 ml of ice-cooled 70% ethanol. Invert the tube and dry at room temperature until the trace ethanol has evaporated. Resuspend the precipitate in 50 μl TE (pH 8.0).
15. For restriction enzyme analysis, digest 5-15 μl (about 1 μg) of resuspended DNA and analyze the product by 0.5% agarose gel electrophoresis or pulsed field gel electrophoresis. 
