Experimental extraction of λ phage DNA from mass cultures using proteinase K and SDS
DNA can be well isolated from large-scale preparations of λ phage by digestion with an effective protease (e.g., proteinase K) and subsequent phenol:chloroform extraction, and this method is also suitable for small-volume (50-100 ml) preparations. This method is also applicable to small volume (50~100 ml ) preparations. This experiment is based on the "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Operation method
Experimental extraction of λ phage DNA from mass cultures using proteinase K and SDS
Principle
DNA can be well isolated from large-scale preparations of λ phage by digestion with an effective protease (e.g., proteinase K) and subsequent phenol:chloroform extraction.This method is also suitable for small (50-100 ml) preparations.
Materials and Instruments
Protease K Restriction endonuclease λ Phage particles Move I. Materials For more product details, please visit Aladdin Scientific website.
Chloroform Dialysis buffer EDTA Ethanol Phenol Phenol: chloroform SDS Sodium acetate TE
Agarose gel Sorvall SS-34 rotor or equivalent Borosilicate Pasteur pipettes Water baths
1. Buffers and solutions
(1) Chloroform
(2) Dialysis buffer
10 mmol/L NaCl
50 mmol/L Tris-Cl (pH 8.0)
10 mmol/L MgCl2
Two portions of dialysis buffer, each 1000 times the volume of the phage solution, are required and stored at room temperature.
(3) EDTA ( 0.5 mol/L, pH 8.0)
(4) Ethanol
(5) Phenol
(6) Phenol: chloroform (1:1, V/V)
(7) SDS (10%, m/V)
(8) Sodium acetate (3 mol/L, pH 7.0)
(9) TE (pH 7.6 and pH 8.0)
2. Enzyme and buffer
Protease K
restriction endonucleases
3. gels
Agarose gel (0.7%) with 0.5X TBE containing 0.5 μg/ml ethidium bromide
4. centrifuge and rotor
Sorvall SS-34 rotor or equivalent
5. Specialized equipment
Borosilicate Pasteur pipette (sealed) or Shepherd hooks
Dialysis bag, boiled
Pre-set 56°C water bath
6. Carriers and strains
λ phage particles in CsCl suspension
II. METHODS
Dialysis of phage suspensions
1. Put the prepared phage suspension into a dialysis bag with one end knotted or closed with a plastic clip, then seal the other end of the bag and put it into a beaker containing more than 1,000 times dialysis buffer and a magnetic stirrer, and dialyze it for 1 h at room temperature with slow stirring.
2. Transfer the bag to another beaker with fresh buffer and dialyze for 1 hour.
3. Transfer the phage suspension into a polypropylene centrifuge tube.
Do not exceed 1/3 of the tube volume.
4. Add 0.5 mol/L EDTA (pH 8.0) to the dialyzed phage suspension to reach a final concentration of 20 mmol/L.
Extraction of phage particles
5. Add proteinase K to the suspension to a final concentration of 50 μg/ml.
6. Add SDS to a final concentration of 0.5% and mix by gently turning the centrifuge tube up and down several times.
The appearance of the solution will rapidly change from a slightly blue suspension of phage particles to a clear solution containing viral DNA and viral capsid protein lysates.
7. Incubate the digestion mixture at 56°C for 1 h and then cool to room temperature.
8. Equal volumes of equilibrium phenol are added and the organic and aqueous phases are mixed by gently turning the centrifuge tube up and down several times until a complete emulsion is formed.
9. Separate the two phases by centrifugation at 3000 g (5000 r/min in a Sorvall SS-34 rotor) for 5 min at room temperature, and transfer the hydrophilic phase to a clean centrifuge tube using a wide-mouth pipette.
10. Extract the hydrophilic phase with a 1:1 mixture of equilibrium phenol and chloroform.
11. Recover the hydrophilic phase as described above (Step 9) and re-extract once with an equal volume of chloroform. For large-scale preparations, proceed to Step 12.
Small volume preparation (phage prepared from 50-100 ml culture)
(1) Recover phage DNA by standard ethanol precipitation.
(2) Leave the solution at room temperature for 30 min.
When ethanol is added, λ phage DNA will appear as a filamentous precipitate, which can be fished out of the solution with a sealed borosilicate Pasteur pipette or Shepherd's hook.
(3) Re-dissolve the DNA in an appropriate volume of TE (pH 7.6) and transfer to step mule 14.
Removal of CsCl
12. Transfer the hydrophilic phase to the dialysis bag.
13. Dialyze the phage DNA sample in 1000x TE (pH 8.0) at 4°C overnight, changing the TE solution three times.
14. Calculate the DNA concentration by measuring the absorbance at 260 nm.
1OD260 = 50 μg/ml of double-stranded DNA. a single phage particle contains approximately 5X10-11 μg of DNA. the yield of phage DNA is dependent on the titer of phage in the lysate culture and is typically 500 μg to several milligrams per liter.
15. The integrity of phage DNA is verified by analysis of undigested or cut portions of DNA (0.5 μg) with appropriate restriction enzymes by agarose electrophoresis at 0.7% using appropriately sized molecular mass standards.
16. Store the phage DNA at 4℃.