DNA extraction using Chelex 100 and proteinase K

Let me start by saying that the following extraction protocol is far from perfect. The product is dirty and often degrades at a faster rate than other extraction methods. Though for my work on insect genetics,  I find this protocol ideal because it is fast and cheap.

The extraction process consists of the addition of proteinase -K, Cheelex 100, and heat. Cheelex is used to protect the DNA from the degrading effects of proteinase K , which is added to free DNA from cells. Heat first speeds up the enzymatic degradation of the cells and then stops the enzymes by degradation. The extraction protocol is as follows:

  1. Prepare samples by placing tissues into 1.5 ml Eppendorf tubes, taking care to allow ethanol to evaporate (if the sample was stored in it).
  2. Add 200ul  of 10 % Chelex 100 per tube.
  3. Crush samples in Chelex 100 (I use melted pipette tips as tiny pestles).
  4. Add 1ul proteinase K (10 mg/ml) to each tube.
  5. Crush samples again.
  6. Vortex tubes for 10 seconds.
  7. Incubate on plate at 57 °C for 1 hour (up to 24 hours).
  8. Vortex for 10 seconds
  9. Boil at 95 °C for 5 minutes.
  10. Vortex for 10 Seconds
  11. Centrifuge at max speed (14,000 RPM) for 10 minutes.
  12. Take supernate and put in new labeled Eppendorf tubes.
  13. Create a working dilution (I usually do 1:10 or 1:50).
  14. Store at 4°C.Photo Oct 21, 10 33 26 AM

One thought on “DNA extraction using Chelex 100 and proteinase K

  1. Hi there!
    I used a similar protocol, but with 2uL of proteinase k (20mg/ml) instead. And also, I boiled it for 10 minutes, not five. The results are pretty suitable for PCR amplifications!

    🙂

    Like

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