Team:Ionis Paris/04 08 16
Purification and quantification of BB123 plasmid (biosensor) extracted from bacterial mini-cultures in order to get this biobrick in stock. 2 Mini-cultures of bacteria transformed with BB123 realized the 03/08 (put a colony with satisfying PCR results in 5 mL LB+Cm into a 50 mL Falcon tube). The miniprep were realized using the Monarch® Plasmid DNA Miniprep Kit (NEB, #T1010) and following the protocol given by the supplier (available here) 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 50 μL Buffer B1 and pool the 4 Eppendorf tubes into a unique tube. Add 200 μL Buffer B2 and mix by inverting the tube 6 times. The solution turns blue. Add 400 μL Buffer B3 and mix by inverting the tube 6 times. The solution turns colorless. Centrifuge for 5 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 200 µL Wash Buffer 1. Centrifuge for 1 min at 13,000 rpm and discard the flow-through. Add 400 µL Wash Buffer 2. 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 Elution Buffer 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. 6 tubes of BB123 (3 per mini-cultures) 2. Store at -80°C. NB: The ratio 260/280 were close to 1.8 meaning that our samples are quite pure.
Miniprep: on DH5⍺ transformed with BB123
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 :
Results
Nanodrop quantification :
