DNA isolation – Proteinase K method

DNA isolation – Proteinase K method

DNA Isolation from Tails

  • Each tail should be in a clean eppendorf tube.
  • Add 500 µl of tail lysis buffer containing proteinase K to each tube.
  • Incubate tail samples in 50-60°C water bath overnight.
  • Add 250 µl saturated (6M) NaCl to each tube.
  • Shake tubes vigorously (~20 times) and incubate tubes on ice for 10 minutes.
  • Spin tubes on low speed (#6 on Hemle centrifuge) at 4°C for 10 minutes.
  • Remove supernatant and place into a clean eppendorf.
  • Add 650 µl isopropanol and invert to mix. Incubate tubes at room temperature for 15 minutes.
  • Recover DNA by centrifuging, max speed, 10 minutes at room temp.
  • Place tubes inverted on bench and allow to air dry 5 minutes.
  • Add 200 µl of TE pH 7.5 or sterile water to each tube. Incubate in 50-60°C water bath for 10 minutes. Re-suspend pellet by pipetting up and down several times.

Tail Lysis Buffer

Proteinase K concentration:
Add 20 µl of a 20 mg/ml stock per 1ml of tail lysis buffer.

Embryonic stem cell (ES Cells)
For ES Cells the protocol is very much the same except for the following: All steps are done in a well of a 24 or 6-well dish.  The initial incubation in the lysis buffer is done at 37°C for 2 hours to overnight.

Southerns:

For important Southerns

  • Dilute DNA in 400µl of water.
  • Phenol/chloroform extract DNA.
  • Precipitate in 1/10 vol 3M NaOAc and equal volume of isopropanol.
  • Precipitate 15 minutes at RT.
  • Wash pellet with 70% EtOH.
  • Resuspend in water.

*We would like to acknowledge & thank the following group: The Jacks Lab. Prof. Jacks is a Howard Hughes Medical Institute Investigator. Our studies are also supported by the National Institutes of Health, the Ludwig Fund for Cancer Research and the Lustgarten Foundation. Prof. Jacks is a Daniel K. Ludwig Scholar and the David H. Koch Professor of Biology at MIT, http://web.mit.edu/jacks-lab/.

Proteinase K Antigen Retrieval Protocol

Description:

Formalin or other aldehyde fixation forms protein cross-links that mask the antigenic sites in tissue specimens, thereby giving weak or false negative staining for immunohistochemically detection of certain proteins. The Proteinase K based solution is designed to break the protein cross-links, therefore unmask the antigens and epitopes in formalin-fixed and paraffin embedded tissue sections, thus enhancing staining intensity of antibodies.

Solutions and Reagents:

  • Proteinase K Solution (20 µg/ml in TE Buffer, pH 8.0)
    TE Buffer (50mM Tris Base, 1mM EDTA, 0.5% Triton X-100, pH 8.0):

    Tris Base – 6.10 g

    EDTA – 0.37 g

    Triton X-100 – 5 ml

    Distilled water – 1000 ml

    Mix to dissolve. Adjust pH 8.0 using concentrated HCl (10N HCl). Store at room temperature.

  • Proteinase K Stock Solution (20x, 400 µg/ml or 12 units/ml)
    Proteinase K (30 units/mg)- 0.008 g (8 mg)

    TE Buffer, pH8.0 – 10 ml

    Glycerol – 10 ml

    Add Proteinase K to TE buffer until dissolved. Then add glycerol and mix well. Aliquot and store at 20°C for 2-3 years.

  • Working Solution (1x, 20 µg/ml or 0.6 units/ml)

    Proteinase K Stock Solution (20x) – 1 ml

    TE Buffer, pH8.0 – 19 ml

    Mix well. This solution is stable for 6 months at 4°C.

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