CsCl iso-density gradient centrifugation is used to prepare the highest purity infectious λ phage pellet, which is free of any bacterial nucleic acid contamination. The DNA extracted from this phage pellet can be used as a template for DNA sequencing, subcloning into plasmid vectors, and generating probes for in situ hybridization. A second advantage of this method is that the phage progeny obtained from it are highly concentrated (>1011 cfu/ml) and retain their infectivity for many years when stored at 4°C. The phage progeny can be used as templates for DNA sequencing, subcloning into plasmid vectors, and generating probes for in situ hybridization. This experiment is based on the "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Operation method
Experimental purification of λ phage particles by isodensity gradient centrifugation with CsCl
Principle
CsCl isodensity gradient centrifugation is used to prepare the highest purity infectious λ phage pellet, which is free of any bacterial nucleic acid contamination. The DNA extracted from this phage pellet can be used as a template for DNA sequencing, subcloning into plasmid vectors, and generating probes for in situ hybridization. A second advantage of this method is that the phage progeny obtained from it are highly concentrated (>1011 cfu/ml) and retain their infectivity for many years when stored at 4°C. The phage progeny can be used as templates for DNA sequencing, subcloning into plasmid vectors and generating probes for in situ hybridization.
Materials and Instruments
λ Phage suspension Move I. Materials For more product details, please visit Aladdin Scientific website.
CsCl Ethanol
Beckman SW41 or SW28 rotor or equivalent Beckman Ti50 or SW50.1 rotor or equivalent Needle for hypodermic injection
1. Buffers and solutions
(1) CsCl (solid)
(2) CsCl solution 
(3) Ethanol
2. centrifuge and rotor
(1) Beckman SW41 or SW28 rotors or equivalent
(2) Beckman Ti50 or SW50.1 rotor or equivalent with clear centrifuge tubes (e.g., Beckman ultra-bright centrifuge tubes)
3. Specialized equipment
Needle for hypodermic injection (21 gauge)
4. Carriers and strains
λ Phage suspension
II. Methods
1. Determine the volume of phage suspension by adding 0.5 g of solid CsCl per ml and stirring and mixing to dissolve it completely.
2. Pour in enough CsCl to make a graded gradient so that the phage suspension is divided into several components, each gradient can hold about 16 ml of phage suspension. The amount of graded gradient required is equal to the final volume of the hydrophilic phase (Step 1), which is divided into fractions of 0.4 X tube volume. Use clear plastic centrifuge tubes (e.g. Beckman Ultra Bright Centrifuge Tubes) suitable for Beckman SW41 or SW28 rotors (or equivalent). 
3. Mark the outside of the corresponding tube wall at the interface between the ρ=1.50 g/ml and ρ=1.45 g/ml layers with a permanent label marker. 
The CsCl gradient is approximately 60% of the volume of the ultra-isolator. For example, for a Beckman SW28 tube (or equivalent), which holds 38 ml, the graded gradient consists of three 7.6 ml CsCl solutions. The equilibrium tube contains the same density of CsCl solution.
4. Carefully add the phage suspension to the graded gradient and centrifuge at 87,000 g (22,000 r/min in a Beckman SW28 rotor) at 4°C for 2 h. The phage suspension is then centrifuged for 2 h at 4°C.
5. Perforate the centrifuge tube as described below to collect the phage pellet.
(1) Carefully wipe off any oil on the outside of the centrifuge tube with alcohol, and then apply Scotch tape to the outside of the centrifuge tube, flush with the phage pellet band.
The tape acts as a seal to prevent leakage around the needle.
(2) A 21-gauge hypodermic needle (no syringe) is inserted through the tape into the centrifuge tube to collect the phage pellet. 
Keep your fingers away from the direction of needle puncture to avoid puncturing the centrifuge tube. Be careful not to contaminate the phage pellet with other bands in the gradient. These bands contain cellular debris and unassembled phage components.
6. Load the phage pellet suspension into an ultra-isolator tube suitable for a Beckman Ti50 or SW50.1 rotor (or equivalent) and add CsCl solution (ρ=1.5 g/ml in SM). centrifuge the pellet at 4°C, 150,000 g (41,000 r/min in a Beckman Ti50 rotor) for 24 h or 160,000 g (36,000 r/min in a Beckman SW50 rotor). Centrifuge at 150,000 g (41000 r/min in Beckman Ti50) for 24 h or 160,000 g (36000 r/min in Beckman SW50.1) for 24 h. 
7. Collect the phage pellet strips as described in step 5 and store them in CsCl solution at 4°C under seal in a test tube.
8. (Optional) If necessary, the phage pellet can be further purified and concentrated by a new round of CsCl equilibrium gradient centrifugation. Transfer the phage suspension to one or more ultra-isolator tubes suitable for a Beckman SW50.1 rotor (or equivalent), add CsCl solution (ρ=1.5 g/ml in SM), and centrifuge at 160,000 g (36,000 r/min in a Beckman SW50.1 rotor) at 4°C for 24 h. When centrifugation is complete, centrifuge the phage pellet in the "Alternative Solution" as described below. When centrifugation is complete, the phage pellet is collected as described in steps 3 and 4 of the "Alternative Scheme" below. 
