Team:Ionis Paris/Notebook/14 10 2016
Purification of BBC45, BBC3, BBX1 plasmids extracted from bacterial mini-cultures.
8 Mini-cultures of bacteria transformed with BBC45, BBC3, BBX1 realized the 13/10 (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. The miniprep were realized using the QIAprep® Spin Miniprep Kit (Qiagen, ref: 27104) 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. 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 to 100 µL of transformed bacteria in a clean 1.5 mL Eppendorf tube. 6 tubes of BBC45 (3 per mini-cultures) 12 tubes of BBC3 (3 per mini-cultures) 12 tubes of BBX1 (3 per mini-cultures)
Miniprep: on DH5⍺ transformed with BBC45/BBC3/BBX1
Objectives
Materials
Protocol
Miniprep:
Bacteria storage :
