Brennan:Miniprep for vaccinia virus DNA isolation

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Contents

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.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

Please sign your name to your note by adding *'''~~~~''': to the beginning of your tip.

  • 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.

References

Relevant papers and books

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

Contact

  • Who has experience with this protocol?

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