Experimental purification of λ phage particles by centrifugation in a graded gradient of glycerol

Summary

This protocol is suitable for the preparation of phage progenitors, the preparation of phage particles (used to obtain DNA for subcloning), and the preparation of λ phage arms. This experiment is from "Guide to Molecular Cloning Experiments, Third Edition" translated by Huang Peitang et al.

Operation method

Experimental purification of λ phage particles by centrifugation in a graded gradient of glycerol

Principle

This protocol is applicable to the preparation of phage progenitors, the preparation of phage particles (used to obtain DNA for subcloning), and the preparation of λ phage arms.

Materials and Instruments

λ Phage particle suspension
EDTA Glycerol SM Tryptic DNase I
Beckman SW41 or SW28 rotor or equivalent Tubes

Move

I. Materials

1. Buffers and solutions

EDTA ( 0.5 mol/L, pH 8.0 )

Glycerol (5% and 40%, V/V ) in SM

SM

2. Enzyme and buffer

Tryptic DNase I (1 mg/ml)

Tryptic RNase in TE (1 mg/ml, pH 7.6)

3. Centrifuge and rotor

Beckman SW41 or SW28 rotor or equivalent with clear centrifuge tubes (e.g., Beckman Ultra Bright Centrifuge Tubes)

4. carriers and strains

λ Phage Pellet Suspension

II. Methods

1. Prepare a glycerol graded gradient (5 ml of phage suspension per tube) in Beckman SW41 polycarbonate centrifuge tubes (or equivalent):

(1) Take 3 ml of SM containing 40% glycerol and add to the bottom of the centrifuge tube.

(2) Carefully add 4 ml of SM containing 5% glycerol to the 40% glycerol solution.

(3) Carefully add the phage suspension to the top layer of 5% glycerol and finally top up with SM.

2. Centrifuge in a graded gradient at 151000 g (35000 r/min in a Beckman SW41 or SW28 rotor) at 4°C for 60 min.

3. Remove the supernatant and resuspend the phage precipitate with 1 ml of SM per liter of culture stock.

4. Add tryptic DNase I and RNase to a final concentration of 5 μg/ml and 1 μg/ml, respectively, and incubate at 37℃ for 30 min.

5. Add 0.5 mol/L EDTA reservoir solution (pH 8.0) to a final concentration of 20 mmol/L.

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