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  1. The cells you recieve will probably be suspended in ethanol. Pipette about 800ul of the cell in ethanol liquid into a microcentrifuge tube. Make sure the centrifuge is balanced, and centrifuge at 300 x g for about 5 min. You should be able to see a white solid at the bottom.
  2. Pipette 200ul of PBS (phosphate buffer saline which may be found in the fridge in the lab) into the microcentrifuge tube with your cells to resuspend them.
  3. Add 20ul proteinase K.
  4. Add 200ul Buffer AL (without ethanol added) and vortex it until it looks mixed and put it in the incubator at 56°C for 10 minutes.
  5. Add 200ul ethanol (96-100%) and vortex until it is homogeneous.
  6. Pipette the mixture (set the pipette to 600ul) into a spin column and make sure to label the cap, and centrifuge it at 8000 rpm for 1 min (you have to set the centrifuge to 2 min, and once it reaches 8000 rpm start your watch and time it for 1 min).
  7. Throw out the collection tube full of liquid and put the spin column into a new tube. Add Buffer 500ul AW1 and centrifuge at 8000 rpm for 1 min.
  8. Throw out the collection tube full of liquid and put the spin column into a new tube. Add Buffer 500ul AW2 and centrifuge at 130 000 rpm for 5 min.
  9. Throw out the collection tube full of liquid and remove the spin column carefully as not to let it touch the liquid and put the spin column into a 1.5 or 1.7 ml microcentrifuge tube (not from the kit, one with a cap) and label it.
  10. Add 100ul Buffer AE (elution buffer) and let it sit at room temperature for 1 min. Centrifuge at 8000 rpm for 1 min.
  11. The liquid in the bottom of the microcentrifuge tube is your DNA.


Return to Manual Extraction.

The next step is the PCR Procedure.


Updated May 3 2009