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DNA isolation - Proteinase K method

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Glycerol, ACS Grade

Buffers > ( G - N ) Buffers

CAS Number:56-81-5

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500 ML $ 37.80
4 L $ 178.20
20 L $ 712.78

DNA Isolation from Tails: Each tail should be in a clean eppendorf tube. DNA Isolation from Tails: Add 500µl of tail lysis buffer containing Proteinase K (PK)to each tube. Incubate tail samples in 50-60C water bath overnight. Add 250µl saturated (6M) NaCl to each tube. ...

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 gEDTA - 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 mlGlycerol - 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 mlTE Buffer, pH8.0 - 19 ml Mix well. This solution is stable for 6 months at 4°C.