Difference between revisions of "Team:Ionis Paris/29 07 16"
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| − | <h4>IONIS | + | <h4>iGEM IONIS</h4> |
| − | <p> We're a group of six different schools from the IONIS Education Group. For this competition we wanted to take advantages of the multiple schools and activity field given by the IONIS education group to create a solid project.</p> | + | <p> We're a group of six different schools from the IONIS Education Group. For this |
| − | + | competition we wanted to take advantages of the multiple schools and activity field | |
| + | given by the IONIS education group to create a solid project.</p> | ||
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| − | <li><a href="https://2016.igem.org/ | + | <li><a href="https://2016.igem.org/Team:Ionis_Paris/Measurement"> Side Projects</a></li> |
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| − | <li><a href="https://2016.igem.org/Team:Ionis_Paris/ | + | <li><a href="https://2016.igem.org/Team:Ionis_Paris/HPintro">Human |
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| − | <p>©IONIS_IGEM_2016</a>.</p> | + | <p>©IONIS_IGEM_2016</a>.</p> |
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Revision as of 12:12, 18 October 2016
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. 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). The miniprep were realized using the QIAprep® Spin Miniprep Kit (Qiagen, ref: 27104) and following the protocol given by the supplier 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. 24 tubes of BB2 3 tubes of BB3 12 tubes of BB12 NB: The ratio 260/280 were close to 1.8 meaning that our samples are quite pure.
Mini prep: on DH5⍺ transformed with BB12, BB2 and BB3
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 :
Protocol
Nanodrop quantification:
