Team:Ionis Paris/22 09 16
Purification and quantification of BBA and BBB plasmids extracted from bacterial mini-cultures in order to sequence them. 28 Mini-cultures of bacteria transformed with BBA (n°1 to 14) and BBB (n°1 to 14) realized the 21/09 (put a colony with satisfying PCR results in 5 mL LB+Cm into a 50 mL Falcon tube). The miniprep were realized using the QIAprep® Spin Miniprep Kit (Qiagen, ref: 27104) and following the protocol given by the supplier (available on thislink). 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 50% to 100 µL of transformed bacteria in clean microcentrifuge 1.5 mL Eppendorf. 42 tubes of BBA (3 per mini-cultures) 42 tubes of BBB (3 per mini-cultures) 2. Store at -80°C.
Mini prep: on DH5⍺ transformed with BBA and BBB
Objectives
Materials
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
Miniprep :
Bacteria storage :
