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 Move I. Materials For more product details, please visit Aladdin Scientific website.
EDTA Glycerol SM Tryptic DNase I
Beckman SW41 or SW28 rotor or equivalent Tubes
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.