Brennan:Miniprep for vaccinia virus DNA isolation
This protocol is to isolate vaccinia virus DNA from one 10cm dish of infected cells.
- 1X PBS, 4°C
- 10% Triton X-100
- 250 mM EDTA, pH 8.0
- Proteinase K (10 mg/mL)
- 3.0 M NaCl
- 10% SDS
- EtOH, 100% and 70%
- 3M NaAcetate
- Rubber policeman
- 15 mL Falcon tubes
- Eppendorf tubes
- Scrape cells from the plate, using a rubber policeman if necessary.
- Transfer cells to a 15 mL Falcon tube and centrifuge at 900g x 10 minutes at 4°C.
- Wash pellet once with 1X PBS (4°C), and repeat step 2.
- Resuspend pellet in 600 μL of PBS and transfer to a 1.5 mL Eppendorf tube.
- To each sample add:
- 30 μL 10% triton X-100
- 1.5 μL β-mercaptoethanol
- 48 μL 250 mM EDTA (pH 8.0)
- Vortex, then incubate on ice 10 minutes while vortexing occassionally (~ every 3 minutes).
- Centrifuge at 700g x 2.5 minutes to remove cellular material.
- Transfer supernatant to a new Eppendorf tube and centrifuge at 16.1K x g for 10 minutes to pellet the viral cores.
- Aspirate supernatant and gently resuspend the pellet in 100 μL Tris-EDTA, pH 8.0.
- To each sample add:
- 1.5 μL proteinase K (10 mg/mL)
- 6.7 μL 3 M NaCl
- 10 μL 10% SDS
- 0.3 μL β-mercaptoethanol
- Mix gently by flicking the tube, and incubate at 55°C for 30 minutes, flicking occasionally (~ every 10 minutes)
Do NOT vortex samples at any point after this line.
- Extract DNA twice with an equal volume of phenol:chloroform.
- Precipitate DNA with 10% volume Na Acetate and 2.5 volumes of 100% EtOH.
- Resuspend pellet in 100 μL Tris-EDTA and repeat the DNA precipitation.
- Wash pellet with 70% EtOH 3x, air dry, then resuspend in 20 μL Tris-EDTA.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
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- Greg Brennan 18:49, 6 May 2015 (EDT) I've used this protocol successfully with as little as a single well from a 6-well plate of starting material.
Relevant papers and books
- Esposito J, Condit R, Obijeski J. (1981) - J Virol Methods. 1981 Feb;2(3) 175-9. PMID 6268651
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