How to do DNA extraction

How to do DNA extraction

Materials

  • Sterile loop
  • PBS buffer
  • AW1 buffer
  • AW2 buffer
  • AE buffer 
  • Proteinase K
  • Ethanol
  • Tube racks
  • Pipette
  • Pipette tips
  • Microfuge tube
  • Spin column
  • Centrifuge
  • Trash bucket

 

 

To perform DNA extraction on a pure colony, collect a tiny amount of cells from the pure colony using a disposable sterile loop. Then resuspend the cells on the sterile loop in 200uL of PBS in a microfuge tube. Next, add 20 uL of proteinase K and gently mix by tapping the tube. Add another 200 uL of ethanol and mix by vortexing. In a 2mL collection tube, place a spin column inside it and pipette the mixture from the microfuge tube in the spin column. Then centrifuge at maximum for 1 min. After 1 min, discard the flow through in the collection tube and place the spin column in a new 2 mL collection tube. Now, add 500 ul of AW1 buffer in the spin column and centrifuge for 1 min at max speed. After another min, discard the flow through again and place the spin column in a new 2 mL collection tube.  Now, using the AW2 buffer, add 500 uL of this buffer in the spin column and centrifuge for 3 min at max. Discard the flow through again in the collection tube and transfer the spin column to a new 1.5 mL microfuge tube. After, elute the DNA in the spin column by adding 200uL of the AE buffer and let sit at room temperature for 1 min. After a min, centrifuge at 1 min at maximum speed, discard the spin column and keep/label the microfuge tube with the DNA of the pure colony inside. 

 

Photo by me is licensed under CC by 2.0

 



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