DNA Extraction

Isolate and extract DNA from crude samples for analysis

Prior to amplification or analysis of DNA, the DNA must be extracted from the tissue sample. A small tissue sample of an organism can be ground and pulverized by hand using a pestle and a micro centrifuge tube. This pulverized sample is now referred to as the crude sample. This process continues by rupturing and denaturing the cell wall (composed of a lipid membrane and proteins), called lysing. Cells are lysed with lysis solution and exposed to elevated temperatures in two water baths, like the Poly Pro Bath or Dual Poly Pro Bath. A micro centrifuge, like a RevSpin and vortexer are also used in the process to remove the lipids and proteins from the mixture. When the process is complete, the DNA chromosomes can be viewed under a microscope and is ready to be amplified in a PCR machine like the RevCycler or analyzed through DNA electrophoresis in a DNA RevBox or even sequenced.

  1. From your samples, obtain a piece of plant or animal tissue approximately 10-20 mg (or ¼ inch diameter if a leaf disc). If you are working with more than one sample, be careful not to cross-contaminate the specimens. (If you only have one specimen, make a balance tube with the appropriate volume of water for the centrifuge step).
  2. Place the sample in a clean 1.5-mL tube labeled with an identification number.
  3. Add 300 uL of lysis solution to the tube.
  4. Twist a clean plastic pestle against the inner surface of the tube to forcefully grind the tissue for 2-5 minutes. Use a clean pestle for each tube if you are doing more than one sample.
  5. Incubate the tube in a Poly Pro Bath at 65C for 10 minutes.
  6. Place your tube in a balanced configuration in a RevSpin microcentrifuge with a cap hinges pointing outward. Centrifuge for 1 minute at maximum speed to pellet debris.
  7. Label a clean 1.5 mL tube with your sample number. Transfer 150uL of the supernatant (clear solution above pellet at bottom of tube) to the clean tube. Be careful not to disturb the debris pellet when transferring the supernatant. Discard the old tube containing the debris.
  8. Add 3uL of silica resin to tube. Mix well by pipetting up and down. Close and incubate the tube in a Poly Pro Water bath at 57C.
  9. Place your tube in a balanced configuration in a RevSpin microcentrifuge. With the cap hinges pointing outward. Centrifuge for 30 seconds at macimum speed to pellet the resin. Use a micropipet with fresh tip to remove the supernatant, being careful not to disrupt the white pellet at the bottom of the tube.
  10. Add 500 uL of ice-cold wash buffer to the pellet. Close the tube and mix well by vortexing or by pipetting up and down to resuspend the silica resin.
  11. Place your tube in a balanced configuration in a RevSpin microcentrifuge. With the cap hinges pointing outward. Centrifuge for 30 seconds at maximum speed to pellet the resin. Use a micropipet with fresh tip to remove the supernatant, being careful not to disrupt the white pellet at the bottom of the tube.
  12. Once again, add 500 uL of ice-cold wash buffer to the pellet. Close the tube and mix well by vortexing or by pipetting up and down to resuspend the silica resin.
  13. Place your tube in a balanced configuration in a RevSpin microcentrifuge. With the cap hinges pointing outward. Centrifuge for 30 seconds at maximum speed to pellet the resin.
  14. Use a micropipet with fresh tip to remove the supernatant, being careful not to disript the white pellet at the bottom of the tube, Spin the tube briefly to collect any drops of supernatant, and rhten remove thses with a micropipet.
  15. Add 100 uL of distilled water to the silica resin and mix well by vortexing or pipetting up and down. Using a Poly Pro Bath, incubate the mixture at 57C for 5 minutes.
  16. Place your tube in a balanced configuration in a RevSpin microcentrifuge. With the cap hinges pointing outward. Centrifuge for 30 seconds at maximum speed to pellet the resin.
  17. Labal a clean 1.5 mL tube with your sample number. Transfer 90 uL of the supernatant to the clean tube. Be careful not to disturb the pellet when transferring the supernatant. Discard the old tube containing the resin.

 

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