Team:Ionis Paris/Notebook/07 10 2016
Amplification of BBX4 plasmid extracted from bacterial mini-cultures in order to have a bigger stock of this plasmid. 1 Mini-culture of bacteria transformed with BBX4 realised the 06/10 (put 100 µL of stock bacteria transformed with BBX4 from 03/10 in 5 mL LB+Cm into a 50 mL Falcon tube).
From this mini-culture, take 300 µL to realize a glycerol stock of tranformed bacteria. The 4.7 mL remaining will serve for the miniprep. The miniprep were realized using the QIAprep® Spin Miniprep Kit (Qiagen, ref: 27104) and following the protocol given by the supplier. Divide the 4.7 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. Resuspend the pellet in 62.5 μL Buffer P1 and pool the 4 Eppendorf tubes into a unique tube. Add 250 μL Buffer P2 and mix by inverting the tube 6 times. The solution turns blue. Add 350 μL Buffer N3 and mix by inverting the tube 6 times. The solution turns colorless. Centrifuge for 10 min at 13,000 rpm. Load 800 μL supernatant from step 5 to the QIAprep 2.0 spin column. Centrifuge for 1 min and discard the flow-through. Add 500 µL Buffer PB. Centrifuge for 1 min at 13,000 rpm and discard the flow-through. Add 750 µL Buffer PE. Centrifuge for 1 min at 13,000 rpm and discard the flow-through. Centrifuge once more for 1 min at 13,000 rpm. Place the QIAprep 2.0 spin column in a clean 1.5 mL microcentrifuge tube. 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. Calculate the quantity of DNA with the Nanodrop. Store the purified DNA at -20°C. 1. Add 100 µL of glycerol 50% to 100 µL of transformed bacteria in clean microcentrifuge 1.5 mL Eppendorf. 3 tubes of BBX4 2. Store at -80°C.
Miniprep: on stock DH5⍺ transformed with BBX4
Objectives
Materials
Protocol
Miniprep :
Bacteria storage :
