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.
