Rapid analysis of λ phage isolates (purification of λ DNA from liquid cultures) Experiments
As described in this scheme, λ phage DNA can be easily purified from liquid cultures. In contrast, the previous scheme describes a method for purifying λ phage DNA from plate lysates. In general, the growth of λ phage in liquid cultures is not as good as that in plate lysates. The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition" translated by Huang Peitang et al.
Operation method
Rapid analysis of λ phage isolates (purification of λ DNA from liquid cultures) assay
Principle
As described in this scheme, λ phage DNA can be easily purified from liquid cultures. In contrast, the previous scheme describes a method for purifying λ phage DNA from plate lysates. In general, the growth of λ phage in liquid cultures is not as good as in plate lysates.
Materials and Instruments
Phage recombinants E. coli Move I. Materials For more product details, please visit Aladdin Scientific website.
Chloroform Ethanol High salt buffer Tris-Cl NaCl EDTA Isopropanol Low salt buffer Phenol: chloroform SM TE TM
NZCYM medium Sorvall SS-34 rotor or equivalent Borosilicate Pasteur pipette Elutip-d column Water bath or heating device Whatman DE52
1. Buffers and solutions
Chloroform
Ethanol
High salt buffer (20 mmol/L Tris-Cl (pH 7.4), 1.0 mol/L NaCl, 1 mmol/L EDTA (pH 8.0))
Isopropyl alcohol
Low salt buffer (20 mmol/L Tris-Cl (pH 7.4), 0.2 moI/L NaCl, 1 mmoI/L EDTA (pH 8.0))
Phenol: chloroform (1:1, V/V)
SM
TE ( pH 8.0)
2. Culture medium
NZCYM medium
3. Centrifuge and rotor
Sorvall SS-34 rotor or equivalent
4. Specialized equipment
Borosilicate Pasteur pipette
Elutip-d column (Schleicher & Schuell)
Pre-set in a 47°C water bath or heating device
Whatman DE52
5. Vectors and strains
Phage recombinants, single phage spots that grow in the moss and are completely independent of each other
Escherichia coli, single completely independent colonies grown on plates
II. Methods
1. A completely independent single phage spot was picked from the plate with a borosilicate pipette, dissolved in 1 ml of SM containing a drop of chloroform, and placed in a small sterile polypropylene tube at 4°C for 4-6 h to allow the phage particles to dissolve out of the top layer of agarose.
2. In a 25 ml centrifuge tube, mix 0.5 ml of phage suspension (about 3X106 phages) with 0.1 ml of overnight cultured bacteria and incubate at 37℃ for 15 min.
3. Add 4 ml of NZCYM medium and incubate at 37℃ with vigorous shaking for about 9 hours. 
4. Add 0.1 ml of chloroform to the culture and continue to incubate at 37℃ with vigorous shaking for 15 min. Transfer the lysate to a 5 ml polypropylene centrifuge tube and centrifuge at 4℃ for 10 min at 800 g (2600 r/min in a Sorvall SS-34 rotor).
5. Transfer the supernatant to a fresh centrifuge tube and centrifuge at 4°C, 4000 g (5800 r/min in a Sorvall SS-34 rotor) for 10 min to remove cellular debris. At this step, a small amount of clear lysate fraction can be set aside as phage stock solution and stored at 4°C with a small amount of chloroform.
6. Take 10 ml of 2:1 DE52 resin suspension and transfer it to a fresh centrifuge tube. Centrifuge at 500 g (2000 r/min in a SorvaIl SS-34 rotor) for 5 min at room temperature to precipitate the resin, remove the supernatant and place the tube in an ice bath.
7. Resuspend DE52 in the clear λ phage supernatant and adsorb the phage pellet onto the resin by shaking the tube for 3 min at room temperature.
8. Centrifuge the λ phage supernatant/DE52 suspension at 4000 g (5800 r/min in a Sorvall SS-34 rotor) for 5 min, carefully transfer the supernatant to another new centrifuge tube and repeat the centrifugation procedure. The sediment was removed after each centrifugation.
9. Transfer the supernatant from the second centrifugation to another new centrifuge tube and extract the supernatant once with phenol: chloroform, which contains the λ phage pellet.
10. Transfer the hydrophilic phase containing λ phage DNA to a new polypropylene centrifuge tube and add an equal volume of isopropanol. Store the mixture at -70°C for 10 min.
11. Collect the precipitated phage DNA by centrifugation at 16,500 g (12,000 r/min in a Sorvall SS-34 rotor) for 20 min at 4 °C.
12. Pour off the isopropanol and let the residue run dry. Air dry the DNA precipitate.
13. Resolve the DNA precipitate with 2 ml of low salt buffer.
14. Purify phage DNA by chromatography using an Elutip-d column.
15. Add 1 ml of ethanol to the DNA eluate, turn the tube upside down several times and ice bath for 20 min. collect the DNA precipitate by centrifugation in a microcentrifuge to remove the supernatant. The DNA precipitate was rinsed with 0.5 ml of 70% ethanol, supernatant was removed, and the DNA was allowed to air dry. The DNA precipitate was dissolved in 50 μl TE (pH 8.0).