Miniprep for vaccinia virus DNA isolation-PDF

Overview

This protocol is to isolate vaccinia virus DNA from one 10cm dish of infected cells.

Materials

Solutions

• 1X PBS, 4°C
• 10% Triton X-100
• β-mercaptoethanol
• 250 mM EDTA, pH 8.0
• Proteinase K (10 mg/mL)
• 3.0 M NaCl
• 10% SDS
• Phenol:chloroform
• EtOH, 100% and 70%
• 3M NaAcetate
• TRIS-EDTA

Equipment

• Rubber policeman
• 15 mL Falcon tubes
• Eppendorf tubes
• Vortex
• Centrifuge

Procedure

1. Scrape cells from the plate, using a rubber policeman if necessary.
2. Transfer cells to a 15 mL Falcon tube and centrifuge at 900g x 10 minutes at 4°C.
3. Wash pellet once with 1X PBS (4°C), and repeat step 2.
4. Resuspend pellet in 600 μL of PBS and transfer to a 1.5 mL Eppendorf tube.
5. To each sample add:
   1. 30 μL 10% triton X-100
   2. 1.5 μL β-mercaptoethanol
   3. 48 μL 250 mM EDTA (pH 8.0)
6. Vortex, then incubate on ice 10 minutes while vortexing occassionally (~ every 3 minutes).
7. Centrifuge at 700g x 2.5 minutes to remove cellular material.
8. Transfer supernatant to a new Eppendorf tube and centrifuge at 16.1K x g for 10 minutes to pellet the viral cores.
9. Aspirate supernatant and gently resuspend the pellet in 100 μL Tris-EDTA, pH 8.0.
10. To each sample add:
    1. 1.5 μL proteinase K (10 mg/mL)
    2. 6.7 μL 3 M NaCl
    3. 10 μL 10% SDS
    4. 0.3 μL β-mercaptoethanol
11. 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.

1. Extract DNA twice with an equal volume of phenol:chloroform.
2. Precipitate DNA with 10% volume Na Acetate and 2.5 volumes of 100% EtOH.
3. Resuspend pellet in 100 μL Tris-EDTA and repeat the DNA precipitation.
4. Wash pellet with 70% EtOH 3x, air dry, then resuspend in 20 μL Tris-EDTA.

References

Relevant papers and books

1. Esposito J, Condit R, Obijeski J. (1981) – J Virol Methods. 1981 Feb;2(3) 175-9. PMID 6268651