Team:Ionis Paris/29 07 16

Mini prep: on DH5⍺ transformed with BB12, BB2 and BB3

Objectives

Purification and quantification of BB12, BB2 and BB3 plasmids extracted from bacterial mini-cultures in order to get these biobricks and use them to construct the whole Biosensor.

Materials

15 Mini-cultures of bacteria transformed with BB2, BB3 or BB12 realized the 28/07 (Put a colony with satisfying PCR results in 5 mL LB+CHL 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

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.

Bacteria storage :

1. Add 100 µL of glycerol to 100 µL of transformed bacteria in a clean 1.5 mL Eppendorf tube.

  • 24 tubes of BB2

  • 3 tubes of BB3

  • 12 tubes of BB12

    • 2. Store at -80°C.

    Protocol

    Nanodrop quantification:

    NB: The ratio 260/280 were close to 1.8 meaning that our samples are quite pure.

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