Kafatos:Minipreps protocol
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Solutions/reagents:
- 5 ml sterile LB medium (+ antibiotic)
- P1 Buffer
- P2 Buffer
- ice-cold P3 Buffer
- absolute EtOH
- 70% ethanol
- TE
- ddH2O
- single bacterial colony
Equipment:
- Incubator
- Centrifuge
- Electrophoretic unit
- Flasks of appropriate volumes
- Eppendorf tubes
Steps:
- DAY 1
Inoculate 5 ml sterile LB medium (+ antibiotic) with single bacterial colony and incubate with shaking for 12 hrs(overnight) at 37°C. - DAY 2
- Measure out 1.5 ml of culture into Eppendorf tube (1).
Centrifuge at a speed of 8000 rpm for 2 mins at room temperature, gently aspirate out the supernatant and discard it. - Add 100 µl of P1 Buffer.
Resuspend P1 Buffer by vortexing/by shaking vigorously.
Store at room temperature for 5 mins. - Add 200 µl of P2 Buffer.
Store at room temperature for at most 5 mins. - Add 150 µl of ice-cold P3 Buffer.
Vortex the mixture for a few secs.
Store the tube on ice for 5 - 10 mins. - Centrifuge at maximum speed for 10 mins at 4°C and aspirate out the top layer.
Transfer top aqueous layer into Eppendorf tube (2).
Discard bottom layer. - Add 2 volumes absolute EtOH to Eppendorf tube (2).
Store the tube on ice for 10 - 20 mins. - Centrifuge at maximum speed for 10 mins at 4°C, gently aspirate out the supernatant and discard it.
- Add 1 ml of 70% ethanol.
Mix solution by pipetting up and down several times. - Centrifuge at maximum speed for 5 mins at room temperature, gently aspirate out the supernatant and discard it.
- Measure out 1.5 ml of culture into Eppendorf tube (1).
Option 1: Dry the pellet in air.
(or)
Option 2: Dry the pellet in speedvac.Do not overdo drying, or the pellet will not properly dissolve again.
Option 1: Add 30 µl of TE.
(or)
Option 2: Add 30 µl of ddH2O.Resuspend ddH2O by vortexing/by shaking vigorously.
Perform agarose gel electrophoresis of appropriate quantity of ddH2O mixed with ethidium bromide and visualize with UV transilluminator to confirm the presence of required product.
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