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− | [[Preparing the pBAD and Lysis plasmids for gel electrophoresis|The Plasmid preparation process]] :
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− | Preparing the pBAD and Lysis plasmids for gel electrophoresis | + | [[Preparing the pBAD and Lysis plasmids for gel electrophoresis|The Plasmid preparation process]] | [[The Plasmid Preparation Miniprep Gallery]] |
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− | 1. Take 1.5µl of the culture in an Eppendorf tube.
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− | 2. Centrifuge the culture @ 13200 rpm for 3 minutes, making sure the centrifuge is balanced. The centrifugal force
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− | created in the centrifuge forces the bacteria in the solution to collect in a pellet at the bottom of the Eppendorf tube.
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− | 3. Repeat process to get required quantity of the cultures i.e. (1.5 µl twice)3.0 µl and centrifuge again.
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− | 4. The liquid around the collected pellet, called the supernatant can be discarded.
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− | 5. Dissolve the pellet in 200µl of ALS I previously stored at 4 degrees Celsius.
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− | 6. Incubate at room temperature(RT) for 3-5 minutes. Incubating at RT enables the DNA to get denatured.
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− | 7. Add 200µl of ALS II - mix properly using a Vortex Mixer and incubate at RT for 5 minutes.
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− | 8. Add 200µl of chilled ALS III, mix properly and incubate the tubes in ice for 10 minutes.
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− | 9. Centrifuge the tubes @ 13.2k rpm for 15 minutes.
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− | 10.Take out the aqueous phase and transfer into a fresh Eppendorf tube.
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− | 11. Add 500µl of Chloroform, mix well and then leave to incubate at RT for 3 minutes.
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− | 12. Now centrifuge the tubes @ 13.2k rpm for 3 minutes.
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− | 13. Take out the upper aqueous phase and transfer into fresh Eppendorf tubes.
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− | 14. Add 0.6 volume of Iso-propanol, incubate @ RT for 15 minutes and then centrifuge @ 13.2 rpm for 20 minutes.
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− | 15. Discard the supernatant and add 500µl of 70% ethanol.
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− | 16. Centrifuge @ 13.2k rpm for 3 minutes at RT and discard the supernatant.
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− | 17. Now add 200µl of absolute Ethanol and centrifuge again for 3 minutes and discard the supernatant.
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− | 18. Dry the pellets at 37-50 degrees Celsius and dissolve them in 20 M1MQ auto-claved (deionized water).
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− | 19. Test the concentrations using a Nanodrop device.
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− | 20. Carry out gel electrophoresis.
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− | Retrieved from "http://www.hackteria.org/index.php/Preparing_the_pBAD_and_Lysis_plasmids_for_gel_electrophoresis"
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− | [[The Plasmid Preparation Miniprep Gallery]]
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