Large-scale preparation of single- and double-stranded M13 phage DNAs
This protocol is mainly used to prepare large quantities of double-stranded DNA from M13 phage, which is often used as a cloning vector in the laboratory, as well as to prepare large quantities of single-stranded phage DNA for specific purposes, such as when a particular recombinant is used multiple times to prepare reflex-labeled probes or to construct large numbers of sentinel mutants. The source of this experiment is the "Laboratory Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Large-scale preparation of single- and double-stranded M13 phage DNA
Principle
This scheme is mainly used to prepare large amounts of double-stranded DNA from M13 phage, which is often used as a cloning vector in the laboratory, as well as to prepare large amounts of single-stranded phage DNA for specific purposes, such as when a particular recombinant is used multiple times to prepare reflex-labeled probes or to construct large numbers of sentinel mutants.
Materials and Instruments
E. coli F' strain M13 Phage stock solution Move I. Materials For more product details, please visit Aladdin Scientific website.
Ethanol NaCl Phenol Chloroform Polyethylene glycol Sodium acetate STE TE Tris-HCl
Agarose gel LB or YT medium Sorvall GSA turn head or equivalent Sorvall SS-34 turn head or equivalent Corex centrifuge tubes Silicon rubber bands Sterile test tubes
1. Buffers and solutions
Ethanol
NaCl (solid)
Phenol
Phenol: chloroform (1:1, V/V)
Polyethylene glycol (20%, m/V, PEG 8000) Soluble in water.
Sodium acetate (3 mol/L, pH 5.2)
STE
TE ( pH 8.0)
Tris-HCl (10 mmol/L, pH 8.0)
2. Gel
Agarose gel (1.2%) suspended in 0.5 X TBE containing 0.5 μg/ml ethidium bromide.
3. Culture medium
5 mmol/L MgCl2 in LB or YT medium
4. centrifuge and rotor
Sorvall GSA turntable or equivalent
Sorvall SS-34 head or equivalent
5. Specialized equipment
Corex tubes (30 ml)
Silicon rubber bands
Sterile tubes (13 mm x 100 mm or 17 mm x 100 mm)
6. Carriers and strains
E. coli F' strain (pick a single colony from a basic M9 agar plate newly isolated with appropriate host bacteria and inoculate into a sterile 20 ml tube containing 5 ml of LB. Incubate at 37°C with gentle shaking for 24-36 h. Do not allow the bacteria to grow to stability as this increases the risk of loss of the F' plasmid-encoded bacteriophage hairs. the single-stranded DNA of the M13 phage, if it is intended to be used as an oligonucleotide-mediated mutation, should be proliferated in E. coli containing mutations in the dut and ung genes).
M13 phage stock solution
II. METHODS
M13 phage RF DNA preparation
1. Transfer 2.5 ml of plate liquid to a sterile test tube (13 X 100 mm or 17 X 100 mm). Add 0.5 ml of M13 phage stock solution (about 5 X 1011 pfu), tap the wall of the tube to mix, and leave at room temperature for 5 min.
2. Dilute the infected cells with 250 ml of LB or YT medium containing 5 mmol/L MgCl2 preheated to 37°C in a 2 L shake flask. incubate for 5 h at 37°C with continuous shaking.
3. Collect the infected cells by centrifugation at 4000 g (Sorvall GSA turntable at 5000 r/min) for 15 min at 4℃. The recovered supernatant can be used for large-scale preparation of single-stranded DNA of M13 phage as described in steps 7 to 17 below.
4. The bacterial precipitate is resuspended in 100 ml of ice-cold STE and the washed cells are recovered by centrifugation at 4000 g at 4°C (Sorvall GSA head at 5000 r/min) for 15 min.
5. Isolate phage closed-loop RF DNA by alkaline lysis. increase the volume of lysate appropriately. Purify the DNA by PEG precipitation or chromatography column purification or CsCI gradient centrifugation.
6. Determine DNA concentration spectrophotometrically and determine integrity by gel electrophoresis. The closed loop DNA is divided into small portions and frozen at -20℃.
The yield of RF DNA from 250 ml of infected cells is approximately 200 μg.
M13 Phage single-stranded DNA preparation
7. Isolate single-stranded DNA from phage particles in the culture medium of infected cells and transfer the supernatant from step 3 to a 500 ml beaker with a magnetic stirrer.
8. Add 10 g of PEG and 7.5 g of NaCl to the supernatant and mix for 30-60 min at room temperature.
9. Centrifuge the supernatant at 10,000 g (Sorvall GSA turntable at 7800 r/min) at 4°C for 20 min to collect the precipitate, invert the flask for 2-3 min to allow the supernatant to drain off, and blot the residual supernatant from the flask wall and neck with a Kimwipes paper towel.
10. 10 ml of 10 mmol/L Tris-HCl (pH 8.0) was added to the bottle, the solution in the bottle was stirred, and the wall of the bottle was rinsed well with a Pasteur pipette, and when the phage precipitate was dissolved, the solution was transferred to a 30 ml Corex centrifuge tube.
11. Add an equal volume of phenol to the phage suspension and shake vigorously for 2 min with a silicone rubber stopper to mix the contents.
12. Centrifuge at 3000 g (Sorvallss-34 turntable at 5000 r/min) for 5 min at room temperature, transfer the upper aqueous phase to a new tube and extract with 10 ml of phenol: chloroform.
13. An equal amount of the aqueous phase is transferred to two 30-ml Corex centrifuge tubes, to which 0.5 ml of 3 mol/L sodium acetate (pH 5.2) and 11 ml of ethanol are added, mixed thoroughly, and allowed to stand at room temperature for 15 min.
14. Centrifuge at 12000 g (Sorvall SS-34 head at 10000 r/min) for 20 min at 4°C to recover the single-stranded DNA precipitate and carefully remove the supernatant.
15. Add 30 ml of 70% ethanol at 4°C to each tube and centrifuge at 12000 g at 4°C (Sorvall SS-34 turntable at 10000 r/min) for 10 min. Carefully remove as much supernatant as possible, invert the tubes to remove any residual liquid from the precipitate, and blot the necks of the tubes with Kimwipes tissue.
16. Allow to stand at room temperature to allow residual ethanol to evaporate, dissolve the precipitate with 1 ml TE (pH 8.0), and store at -20 °C.
17. Determine the DNA concentration spectrophotometrically and determine its integrity by agarose electrophoresis, and divide the closed loop DNA into small portions (10-50 μg) and store at -20℃.