Difference between revisions of "Team:Ionis Paris/29 07 16"
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| − | + | <h2 class="blog_topHd"> <font color =”#279AD3”>Mini prep: on DH5⍺ transformed with BB12, BB2 and BB3</font></h2> | |
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| − | + | <h3><font color =”94FAF1”> Objectives </font></h3> | |
<p>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.</p> | <p>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.</p> | ||
| − | < | + | <h3><font color =”94FAF1”> Materials </font></h3> |
<p>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).<br/> | <p>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).<br/> | ||
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.</p> | 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.</p> | ||
| − | < | + | <h3><font color =”94FAF1”> Protocol </font></h3> |
<p>The miniprep were realized using the QIAprep® Spin Miniprep Kit (Qiagen, ref: 27104) and following the protocol given by the supplier </p> | <p>The miniprep were realized using the QIAprep® Spin Miniprep Kit (Qiagen, ref: 27104) and following the protocol given by the supplier </p> | ||
| − | + | <h5><font color =”#3CB5E1”>Miniprep:</font></h5> | |
<ol> | <ol> | ||
<li><p>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.</p></li> | <li><p>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.</p></li> | ||
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| − | < | + | <h5><font color =”#3CB5E1”>Bacteria storage :</font></h5> |
<p>1. Add 100 µL of glycerol to 100 µL of transformed bacteria in a clean 1.5 mL Eppendorf tube.</p> | <p>1. Add 100 µL of glycerol to 100 µL of transformed bacteria in a clean 1.5 mL Eppendorf tube.</p> | ||
<li><p>24 tubes of BB2</p></li> | <li><p>24 tubes of BB2</p></li> | ||
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2. Store at -80°C. | 2. Store at -80°C. | ||
| − | + | <h3><font color =”94FAF1”> Protocol </font></h3> | |
| − | <h3>Nanodrop quantification:</ | + | <h5><font color =”#3CB5E1”>Nanodrop quantification:</font></h5> |
| − | <figure | + | <figure> |
<img src="https://static.igem.org/mediawiki/2016/9/9a/T--Ionis_Paris--Notebook29_07Fig.1.png" alt=""> | <img src="https://static.igem.org/mediawiki/2016/9/9a/T--Ionis_Paris--Notebook29_07Fig.1.png" alt=""> | ||
</figure> | </figure> | ||
Latest revision as of 21:20, 19 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:
