Preparing the pBAD and Lysis plasmids for gel electrophoresis

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1. Take 1.5µl of the culture in an Eppendorf 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 Eppendorf 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.

11. Add 500µl of Chloroform, mix well and then leave to incubate at RT for 3 minutes.

12. Now centrifuge the tubes @ 13.2k rpm for 3 minutes.

13. Take out the upper aqueous phase and transfer into fresh Eppendorf tubes.

14. Add 0.6 volume of Iso-propanol, incubate @ RT for 15 minutes and then centrifuge @ 13.2 rpm for 20 minutes.

15. Discard the supernatant and add 500µl of 70% ethanol.

16. Centrifuge @ 13.2k rpm for 3 minutes at RT and discard the supernatant.

17. Now add 200µl of absolute Ethanol and centrifuge again for 3 minutes and discard the supernatant.

18. Dry the pellets at 37-50 degrees Celsius and dissolve them in 20 M1MQ auto-claved (deionized water).

19. Test the concentrations using a Nanodrop device.

20. Carry out gel electrophoresis.

Notes- There should not be too much salt in the digestion because too salt salt will inhibit enzyme activity.


        Plasmid   Concentration   260/280
        IO500(P)   1790.5µg/µL     1.92
        Lysis(L)   2324.4µg/µL     1.91

For Short Digestion

                DNA = 2µL
 ECORI + Pst1 E1+E2 = 0.5µL + 0.5µL
                BSA = 2µL
             Buffer = 2µL
                 MQ = 13µL
              Total = 20µL

Time taken = 1.5 hours