Preparing the pBAD and Lysis plasmids for gel electrophoresis

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1. Take 1.5µl of the culture in an Eppendorff tube.

2. Centrifuge the culture @ 13200 rpm for 3 minutes, making sure the centrifuge is balanced.
The centrifugal force created in the centrifuge forces the bacteria in the solution to collect in a pellet at the bottom of the Eppendorff tube.

3. Repeat process to get required quantity of the cultures i.e. (1.5 µl twice)3.0 µl and centrifuge again.

4. The liquid around the collected pellet, called the supernatant can be discarded.

5. Dissolve the pellet in 200µl of ALS I previously stored at 4 degrees Celsius.

6. Incubate at room temperature(RT) for 3-5 minutes. Incubating at RT enables the DNA to get denatured.

7. Add 200µl of ALS II - mix properly using a Vortex Mixer and incubate at RT for 5 minutes.

8. Add 200µl of chilled ALS III, mix properly and incubate the tubes in ice for 10 minutes.

9. Centrifuge the tubes @ 13.2k rpm for 15 minutes.

10.Take out the aqueous phase and transfer into a fresh Eppendorf tube.



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