Mike Barnkob:Protocols/Bacterial/Isolate DNA
Isolate DNA from bacteria
Protocol to isolate bacterial plasmids from overnight liquid cultures.
- QIAprep Spin Miniprep Kit.
- Autoclaved 1.5 mL Eppendorf tubes.
Follows QIAprep Spin Miniprep protocol. Presumes all reagens have been prepared.
Note: All spins conducted at 13.000 RPM.
- Transfer bacterial culture into 2ml tubes.
- Spin for 3 min in mini-centrifuge.
- Remove supernatant and resuspend pellet in 250 μL Buffer P1 (kept at 4°C).
- Add 250 μL Buffer P2 and mix by inverting tubes 6-10 times, until solution turns blue.
Note: Finish this step in under 5 min.
- Add 350 μL Buffer N3 and mix by inverting tubes 6-10 times, until entire solution turns colorless.
- Spin for 10 min in mini-centrifuge.
- Transfer 750 μL of supernatant to spin column. Let sit for 60 sec minimum.
- Spin spin column for 45 seconds. Discard the flow-through by decanting.
- Add 500 μL Buffer PB and spin for 45 seconds. Discard the flow-through by decanting.
- Add 750 μL Buffer PE and spin for 45 seconds. Discard the flow-through by decanting.
- Transfer spin column to 1.5ml Eppendorf tube.
- Spin for 1 min to remove residual wash buffer.
- Transfer spin column to new 1.5ml Eppendorf tube.
- Add 50 μL Buffer EB (10 mM TrisCL) to the center of the spin column, very close to membrane. Let sit for 60 seconds minimum.
- Spin for 1 min. Flow-through is your DNA.
- Label tubes and store at -20°C
- Add 1.2 μL of DNA to NanoDrop and record concentration, 260/280 and 260/230 ratios.
- QIAprep Spin Miniprep Kit protocol.