Rapid analysis of λ phage isolates (purification of λ DNA from plate lysates) assay
Analysis of promising genomic or cDNA clones usually begins with digestion of small preparations of λ phage DNA with restriction enzymes and analysis of the digested products by agarose gel electrophoresis. The resulting DNA can be used as a substrate for restriction enzymes, as a template for RNA synthesis (by DNA-dependent RNA polymerases encoded by phages SP6, T3, and T7), and as a template for PCR. This experiment is based on the "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 plate lysates) assay
Principle
Analysis of promising genomic or cDNA clones usually begins with digestion of small preparations of λ phage DNA with restriction enzymes and analysis of the digested products by agarose gel electrophoresis. The resulting DNA can be used as a substrate for restriction enzymes, as a template for RNA synthesis (by DNA-dependent RNA polymerases encoded by phages SP6, T3, and T7), and as a template for PCR.
Materials and Instruments
λ phage recombinants E. coli spread plate bacteria 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
LB or NZCYM agarose plates LB or NZCYM top agarose 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)
TM
2. Culture medium
LB or NZCYM agarose plates
LB or NZCYM Top Agarose
3. Centrifuges and rotors
Sorvall SS-34 rotor or equivalent
4. Specialized equipment
Borosilicate Pasteur pipette
Elutip-d column (Schleicher & Schuell)
Water bath or heating device preset at 47°C
Whatman DE52
5. Vectors and strains
λ phage recombinants, single, completely independent phage spots grown on the bacterial moss
E. coli spread plate bacteria
II. METHODS
Preparation of lysates
1. Use borosilicate pipette to pick 8~10 completely independent phage spots from the plate which are all derived from a genetically pure, phage-purified phage progenitor species. The phage spots were dissolved in 1 ml of SM and 50 μl of chloroform and placed at 4°C for 4-6 h to dissolve the phage particles from the top layer of agarose.
2. Mix 50~100 μl of phage suspension (about 105 pfu ) with 150 μl of plate-laying bacteria in a small sterile culture tube, incubate at 37℃ for 20 min, add 7.0 ml of melted (47℃) top agarose (0.7%), and pour it onto freshly-filled 150 mm NZCYM agarose plates for spreading culture.
3. Plates were incubated inverted at 37°C until the phage plaques covered almost the entire plate surface (7~9 h).
4. Add 7 ml of TM directly onto the surface of the top layer of agarose and incubate for 4 h at 4 ℃ with gentle continuous shaking to elute the phage particles.
5. Transfer the λ phage eluate to a centrifuge tube and centrifuge at 4000 g (5800 r/min in Sorvall SS-34 rotor) for 10 min at 4°C to remove cellular debris. At this step, a small amount of clear lysate fraction can be retained as phage stock solution, which can be 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 centrifuge tube. Centrifuge at 500 g (2000 r/min in a Sorvall 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 clear TM and adsorb the phage pellet onto the resin by shaking the tube for 3 min at room temperature.
8. Centrifuge the TM/DE52 suspension at 4000 g (5800 r/min in a Sorvall SS-34 rotor) for 5 min, carefully transfer the supernatant to another new tube and repeat the centrifugation procedure. The sediment was removed after each centrifugation.
9. Transfer the supernatant from the second centrifugation to a 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 Ehitip-d column:
(1) Push 1~2 ml of high-salt buffer onto the Elutip-d column with a syringe.
(2) Push 5 ml of low salt buffer onto the column.
(3) Attach a 0.45 μm prefilter to the column and slowly push the DNA sample into the column (Step 13).
(4) Wash the column with 2-3 ml of low salt buffer.
(5) Remove the prefilter and elute the DNA with 0.4 ml of high salt buffer; at this step, collect the eluate in a 1.5 ml microcentrifuge tube.
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 to remove the supernatant and then allowed to air dry. The DNA precipitate was dissolved in 50 μl TE (pH 8.0). 
