If the purpose of purification of the phage pellet is to obtain outgrown DNA fragments for subsequent subcloning or to prepare phage two-arms, centrifugation can be used instead of CsCl graded gradients and equilibrium gradients, and the phage pellet can be deposited at the bottom of the centrifuge tube by centrifugation. Phages obtained by this method are not as pure as those obtained by equilibrium gradient centrifugation or glycerol gradient centrifugation, but they are perfectly adequate for DNA extraction. The source of this experiment is "Molecular Cloning Laboratory Guide, Third Edition", translated by Huang Peitang et al.

Operation method

Experimental purification of λ phage particles by precipitation/centrifugation

Principle

If the purpose of purification of the phage pellet is to obtain outgrown DNA fragments for subsequent subcloning or to prepare phage two-arms, centrifugation can be used instead of CsCl graded gradients and equilibrium gradients, and the phage pellet can be deposited at the bottom of the centrifuge tube by centrifugation. The phage obtained by this method are not as pure as those obtained by equilibrium gradient centrifugation or glycerol gradient centrifugation, but they are perfectly adequate for DNA extraction.

Materials and Instruments

λ Phage Particle Suspension
SM
Beckman SW28 rotor or equivalent

Move

I. Materials

1. Buffers and solutions

SM

2. Centrifuges and rotors

Beckman SW28 rotor or equivalent

3. vectors and strains

λ Phage particle suspension

II. Methods

1. Transfer the phage suspension to a centrifuge tube suitable for Beckman SW28 rotors (or equivalent).

2. Collect the phage pellet by centrifugation at 110,000 g (25,000 r/min in a Beckman SW28 rotor) at 4°C for 2 h. The phage pellet should be collected by centrifugation at 4°C.

3. Carefully pour off the supernatant.

A clear (glassy) phage pellet precipitate can be seen at the bottom of the centrifuge tube.

4. Add 1-2 ml of SM to the precipitate and place at 4°C overnight, preferably on a slowly rotating shaker.

5. The next morning, gently aspirate the solution up and down to ensure that all phage particles have been resuspended.

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