Objectives

Purification and quantification of BB12mut and BB123mut plasmids extracted from bacterial mini-cultures in order to sequence them.

Materials

16 Mini-cultures of bacteria transformed with BB12 (3, 4, 5, 7, 8, 9, 11, 12) and BB123 (1, 2, 4, 5, 7, 10, 11, 13) realized the 06/09 (put a colony with satisfying PCR results in 5 mL LB+Cm into a 50 mL Falcon tube).
From those mini-cultures, take 500 µL to realize a glycerol stock of tranformed bacteria. The 4.5 mL remaining will serve for the miniprep.

Protocol

The miniprep were realized using the QIAprep® Spin Miniprep Kit (Qiagen, ref: 27104) and following the protocol given by the supplier (available here).

Miniprep:

1. Divide each 4.5 mL bacterial O/N mini-cultures into 4 Eppendorf tubes and centrifuge all those tubes at 9,000 rpm for 3 min at room temperature. Discard the supernatant.

2. Resuspend the pellet in 62.5 μL Buffer P1 and pool the 4 Eppendorf tubes into a unique tube.

3. Add 250 μL Buffer P2 and mix by inverting the tube 6 times. The solution turns blue.

4. Add 350 μL Buffer N3 and mix by inverting the tube 6 times. The solution turns colorless.

5. Centrifuge for 10 min at 13,000 rpm.

6. Load 800 μL supernatant from step 5 to the QIAprep 2.0 spin column. Centrifuge for 1 min and discard the flow-through.

7. Add 500 µL Buffer PB. Centrifuge for 1 min at 13,000 rpm and discard the flow-through.

8. Add 750 µL Buffer PE. Centrifuge for 1 min at 13,000 rpm and discard the flow-through.

9. Centrifuge once more for 1 min at 13,000 rpm.

10. Place the QIAprep 2.0 spin column in a clean 1.5 mL microcentrifuge tube.

11. Add 50 μL Buffer EB to the center of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min at 13,000 rpm.

12. Calculate the quantity of DNA with the Nanodrop.

13. Store the purified DNA at -20°C.

Miniprep:

1. Add 100 µL of glycerol 50% to 100 µL of transformed bacteria in clean microcentrifuge 1.5 mL Eppendorf.

   • 32 tubes of BB12mut (4 per mini-cultures)

   • 32 tubes of BB123mut (4 per mini-cultures)

2. Store at -80°C.

   NB: No nanodrop quantification of samples was performed due to the departure of the machine.

   

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