M13 Preparation of phage double-stranded (replicative) DNA
Bacteria infected with M13 phage contain viral double-stranded RF DNA, and single-stranded daughter DNA is contained in crude viral particles in the culture medium. Double-stranded RF DNA can be isolated from small cultures of infected cells using a method similar to that used for plasmid purification. A few micrograms of RF DNA can be isolated from 1 to 2 ml of infected cell culture, and this amount is sufficient for subcloning and restriction enzyme digestion. The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Operation method
M13 Preparation of phage double-stranded (replicative) DNA
Principle
Bacteria infected with M13 phage contain viral double-stranded RF DNA, and single-stranded daughter DNA is contained in crude viral particles in the culture medium. Double-stranded RF DNA can be isolated from small cultures of infected cells using a method similar to that used for plasmid purification. A few micrograms of RF DNA can be isolated from 1 to 2 ml of infected cell culture, an amount sufficient for subcloning and restriction enzyme digestion.
Materials and Instruments
Restriction endonuclease E. coli culture Move I. Materials For more product details, please visit Aladdin Scientific website.
Alkaline lysate Ethanol Phenol Chloroform TE
Agarose gel
1. Buffers and solutions
Alkaline lysis solution Ⅰ
Alkali lysis solution Ⅱ
Alkaline cleavage solution Ⅲ
Ethanol
Phenol: chloroform (1:1, V/V)
TE containing 20 μg/ml Rnase A ( pH 8.0).
2. Enzyme and buffer
Restriction endonuclease
3. gels
Agarose gel (0.8%) suspended in 0.5 X TBE containing 0.5 μg/ml ethidium bromide.
4. Vectors and strains
E. coli culture infected with M13 phage
II. METHODS
Lysis of infected cells
1. 1 ml of M13 infected cell culture was centrifuged at maximum speed for 5 min at room temperature on a microcentrifuge to isolate infected cells from the culture broth. Transfer the supernatant to a new microcentrifuge tube and store at 4°C. The infected cell precipitate was placed on ice.
2. Centrifuge the cell precipitate at 4°C for 5 s and remove the residual culture medium with an automatic pipette.
3. Add 100 μl of pre-chilled Alkaline Lysis Solution I and shake vigorously to suspend the cell precipitate.
4. Add 200 μl of newly configured Alkaline Lysis Solution II to the tube. Cover tightly and mix 5 times with rapid inversion. Do not shake. Place on ice for 2 min after mixing.
5. Add 150 μl of ice-cold Alkaline Lysis Solution III to the tube. Cover tightly and mix upside down several times to mix the Lysate III with the viscous bacterial lysate and place on ice for 3-5 min.
6. On a microcentrifuge, centrifuge the bacterial lysate at maximum speed at 4°C for 5 min and transfer the supernatant to a new microcentrifuge tube.
M13 Purification of phage RF DNA
7. Add equal volumes of phenol: chloroform. Mix the organic and aqueous phases by shaking and centrifuge at maximum speed for 2-5 min. Transfer the aqueous phase (upper phase) to a new microcentrifuge tube.
8. Add 2 times the volume of ethanol to precipitate double-stranded DNA. mix with shaking and leave for 2 min at room temperature.
9. Recover the DNA by centrifugation in a microcentrifuge at 4°C for 5 min at maximum speed.
10. Gently aspirate the supernatant and place the centrifuge tube upside down on blotting paper to drain the tube and remove any droplets adsorbed on the wall of the tube. 
11. Add 1 ml of 70% ethanol and centrifuge at 4°C for 2 min. Remove the supernatant as in step 10 and dry the nucleic acid precipitate at room temperature for 10 min.
12. To remove traces of RNA, the precipitate is resuspended in RNase-containing TE (pH 8.0) with slight shaking.
13. Digest with appropriate restriction endonuclease and analyze double-stranded RF DNA by agarose gel electrophoresis.