M13 phage single-stranded DNA was prepared from viral particles secreted by infected cells into the surrounding culture medium. filoviruses were first concentrated by polyethylene glycol (PEG) precipitation in the presence of high salt, followed by phenol extraction to release the single-stranded DNA, and finally ethanol precipitation to collect the single-stranded DNA. this experiment was derived from the "Guide to molecular cloning experiments, third edition", translated by Huang, Peitang et al.
Operation method
Preparation of M13 phage single-stranded DNA
Principle
M13 Phage single-stranded DNA was prepared from viral particles secreted by infected cells into the surrounding culture medium, firstly in the presence of high salt, filoviruses were concentrated by polyethylene glycol (PEG) precipitation, followed by phenol extraction to release the single-stranded DNA, and finally ethanol precipitation to collect the single-stranded DNA.
Materials and Instruments
M13 single-stranded phage vector Cultures of E. coli infected with M13 phage Cultures of uninfected E. coli Move I. Materials For more product details, please visit Aladdin Scientific website.
Chloroform Ethanol Phenol Polyethylene Glycol 8000 Sodium Acetate Sucrose Gel Sampling Buffer TE
Agarose gel Multi-tube shaker
1. Buffers and solutions
Chloroform
Ethanol
Phenol
Polyethylene glycol 8000 ( 20%, m/V, PEG 8000) was dissolved in 2.5 mol/L NaCl.
Sodium acetate (3 mol/L, pH 5.2)
Sucrose Gel Sampling Buffer
TE ( pH 8.0)
2. Gel
Agarose gel (1.2%) suspended in 0.5 X TBE containing 0.5 μg/ml ethidium bromide.
3. Nucleic acids and oligonucleotides
M13 single-stranded phage vector for recombinant DNA
4. Specialized equipment
Multi-tube oscillator (optional)
5. Carriers and strains
E. coli cultures infected with M13 phage
Cultures of uninfected E. coli
II. METHODS
PEG precipitation of phage particles
1. 1 ml of infected and uninfected cultures were added to two microcentrifuge tubes and centrifuged at maximum speed for 5 min at room temperature, and the supernatants were transferred to two new microcentrifuge tubes.
2. Add 200 μl of 20% PEG dissolved in 2.5 mol/L NaCl to the supernatant, mix the solution by inverting the tubes several times, shake gently, and leave at room temperature for 15 min.
3. Centrifuge at 4℃ for 5 min at maximum speed in a microcentrifuge to recover the phage pellet precipitate.
4. Remove all supernatant by careful aspiration with a disposable pipette tip attached to a vacuum device or a long Pasteur pipette with a rubber bulb and centrifuge for 30 s to remove any residual supernatant.
Phenol extraction of single-stranded DNA
5. Resuspend the phage pellet precipitate with 100 μl TE (pH 8.0) by shaking.
The best way to completely resuspend the precipitate is to soak the precipitate in TE for 15-30 min at room temperature, followed by low speed shaking to dissolve the precipitate. Complete resuspension of the phage pellet precipitate is important for efficient extraction of single-stranded DNA with phenol in the next step, and if multiple samples are being extracted at the same time, the use of a multi-tube oscillator saves time and effort.
6. Add 100 μl of equilibrium phenol to the precipitate suspension, shake for 30 s to mix thoroughly, let stand at room temperature for 1 min, and shake again for 30 s.
7. centrifuge in a microcentrifuge at maximum speed for 3-5 min. transfer as much of the upper aqueous phase as convenient to a new microcentrifuge tube.
To facilitate phase separation, add 30 μl of silicon lubricant. This step sometimes improves the yield, but is usually not necessary. Do not attempt to transfer all of the aqueous phase; single-stranded DNA is cleaner when approximately 5 μl of the aqueous phase remains at the interface.
Templates prepared with a single phenol extraction can be used for most purposes (including DNA sequencing) and generally do not need to be phenol-extracted again. However, if the fraction with FEG/NBa supernatant is contaminated by phage precipitation at Step 3, it can affect the reproducibility of the double deoxy sequencing reaction and reduce the reliable length of each reaction assay to 300 bp or less. Special attention to removing trace supernatants from microcentrifuge tubes can avoid this problem. Some researchers have also added 100 μl of chloroform to each centrifuge tube before centrifugation in step 6 and shaking to mix, or transferred the aqueous phase to a new microcentrifuge tube in step 7, and then extracted the aqueous phase once with 100 μl of chloroform, centrifuged to separate the phases, and transferred the aqueous phase to a new microcentrifuge tube.
Ethanol precipitation of phage DNA
8. Recover M13 DNA by standard ethanol precipitation in the presence of 0.3 mol/L sodium acetate. mix with brief shaking and leave at room temperature for 15-30 min or overnight at -20℃.
The aqueous phenol phase transferred to a new centrifuge tube may be slightly cloudy, but should be clear upon addition of sodium acetate.
Single-stranded DNA can also be precipitated by adding 300 μl of a 25:1 mixture of anhydrous ethanol:3 mol/L sodium acetate and leaving at room temperature for 15 min. This method eliminates the need for separate additions of sodium acetate and ethanol, and thus speeds up the process for many single-stranded DNA sample purifications.The M13 DNA can be stored in ethanol at -20°C for several months.
9. Recover the single-stranded phage DNA precipitate by centrifugation in a microcentrifuge at 4°C for 10 min at maximum speed.
10. Gently aspirate the supernatant, being careful not to touch the DNA precipitate (often only a faint trace can be seen on the wall of the tube), and centrifuge for 15 s to remove any residual supernatant.
11. add 200 μl of cold 70% ethanol and centrifuge at 4°C for 5-10 min at maximum speed. gently aspirate the supernatant immediately.
Important: In this step, the precipitate does not stick to the wall of the tube, so it is important to work quickly and carefully to avoid DNA loss.
12. Invert the uncapped tube for 10 min to allow all residual ethanol to escape and evaporate. Dissolve the precipitate with 40 μl TE (pH 8.0) and incubate at 37°C for 5 min to accelerate DNA dissolution. store the DNA solution at -20°C.
The yield of single-stranded DNA is usually 5-10 μg/ml of the original infected culture.
13. The DNA concentration of several samples can be estimated by mixing 2 μl of the DNA solution prepared in step 12 with 1 μl of Sucrose Gel Spiking Buffer for each sample and adding it to the spiking wells of a 1.2% agarose gel containing 0.5 X TBE and 0.5 μg/ml ethidium bromide. Different amounts of M13 DNA of known concentration were used as a control. 6 V/cm electrophoresis was performed for 1 h, and the amount of DNA was estimated according to the intensity of fluorescence density.
Each set of four double deoxy sequencing reactions using dye-labeled primers typically requires 2 to 3 μg of standard M13 phage DNA product.