If extracted poxvirus DNA is used for transfection, it should be isolated after proteinase K digestion and phenol extraction. See this experiment for specific methods. Source Compact Molecular Biology Laboratory Guide (Fifth Edition)

Operation method

basic program

Materials and Instruments

Purified poxvirus
Tris-Cl solution SDS Sucrose solution Protease K Phenol equilibrated with 50 mmol/L Tris - Cl solution 1:1 phenol chloroform 1 mol/L sodium acetate Ethanol
Spectrophotometer Sorvall centrifuge

Move

1. Determine the optical density of the purified jaundice virus at 260 nm by adding 20 optical density units of the virus (see "Poxvirus Purification Experiment" Step 24) to 50 mmol/L Tris-C buffer pH 7.8, in a final volume of 1.2 ml. 2.

2. Add the following solution to the virus suspension (final volume 2 ml):

0.1 ml 1 mol/L pH 7.8 Tris - Cl

0.1 ml 10 % SDS

0.2 ml 60% sucrose solution

0.4 ml 10 mg/ml Protease K

incubate at 37℃ for 4 h. 3.

3. extract twice with phenol, each time adding an equal volume of equilibrium phenol, shake the centrifuge tube and mix gently, centrifuge at 300 g for 10 min at room temperature, and aspirate the upper aqueous phase with the tip removed.

4. extract once more with 1:1 phenol-chloroform using the method described in step 3. 5. add 1/10 volume of phenol-chloroform.

5. Add 1/10 volume of 1 mol/L sodium acetate pH 7.0 and 2.5 volumes of 100% ethanol. Mix gently and refrigerate at -20°C for several hours. 6.

6. Microcentrifuge at 4°C for 10 min at maximum speed and discard the supernatant. 7.

7. Wash the precipitate twice with 95% ethanol, adding ethanol to each addition and microcentrifuging at maximum speed to aspirate the supernatant. Air dry and dissolve in 100 μl of water. 8.

8. Prepare a dilution (a small portion only) and determine the DNA concentration by _A260.

Caveat

Do not vortex the DNA in this operation because the genome of poxviruses is large and must be handled carefully to avoid shearing the DNA if you want to obtain full-length DNA (e.g., for restriction enzyme digestion analysis).

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