Difference between revisions of "Team:Ionis Paris/09 06 16"
| Line 35: | Line 35: | ||
<div class="bloggrid_right"> | <div class="bloggrid_right"> | ||
<div class="blog_top"> | <div class="blog_top"> | ||
| − | + | <h2 class="blog_topHd"> <font color =”#279AD3”> Competent cells: E.Coli BL21</font></h4> | |
| − | + | ||
</div> | </div> | ||
| − | + | <h3><font color =”94FAF1”> Objectives </font></h3> | |
<p>The objective here is to make a stock of E.Coli BL21 competent cells for subsequent transformations.</p> | <p>The objective here is to make a stock of E.Coli BL21 competent cells for subsequent transformations.</p> | ||
| − | + | <h3><font color =”94FAF1”> Materials </font></h3> | |
| − | + | <h5><font color =”#3CB5E1”> Stock concentrations:</font></h5> | |
<li><p>O/N BL21 pre-culture(made on the 08/06): O/N inoculation of 100 µL BL21 into 100 mL LB.</p> | <li><p>O/N BL21 pre-culture(made on the 08/06): O/N inoculation of 100 µL BL21 into 100 mL LB.</p> | ||
| Line 51: | Line 50: | ||
</li> | </li> | ||
| − | + | <h3><font color =”94FAF1”> Protocol </font></h3> | |
| − | + | <h5><font color =”#3CB5E1”> Competence:</font></h5> | |
<ol> <li><p>Inoculation of 3 mL O/N culture in 100 mL LB in 500 mL erlenmeyer.</p> | <ol> <li><p>Inoculation of 3 mL O/N culture in 100 mL LB in 500 mL erlenmeyer.</p> | ||
| Line 80: | Line 79: | ||
</ol> | </ol> | ||
| − | + | <p><font color ="A6A6A6">NB: The competency of the prepared cells will be tested the 10/06.</font></p> | |
<div role="tabpanel" class="tab-pane fade" id="popular_post"> | <div role="tabpanel" class="tab-pane fade" id="popular_post"> | ||
Revision as of 08:54, 19 October 2016
The objective here is to make a stock of E.Coli BL21 competent cells for subsequent transformations. O/N BL21 pre-culture(made on the 08/06): O/N inoculation of 100 µL BL21 into 100 mL LB. 0.1M CaCl2: prepared on the 05/06 0.1M CaCl2/15% Glycerol: prepared on the 05/06 Inoculation of 3 mL O/N culture in 100 mL LB in 500 mL erlenmeyer. Incubation at 250 rpm at 37°C until the DO 0,6 —> DO = 0.615 Cells were transferred to 3 sterile ice-cold 50 mL Falcon tubes. 20 mL in each falcon tube. Incubate on ice for 30 min. Do not allow cells to warm up over 4°C. Spin cells at 4000 rpm for 10 min at 4°C. Discard supernatant and try to drain all remaining media. Gently resuspend on 10 mL cold 0.1M CaCl2 Incubate on ice 20 min Centrifuge 10 min at 4,000 rpm at 4°C Discard supernatant and gently resuspend on 5 mL cold 0.1M CaCl2/25% Glycerol Transfer in 1.5 mL eppendorf (100 µL/tube) Store at -80°C NB: The competency of the prepared cells will be tested the 10/06.
Competent cells: E.Coli BL21
Objectives
Materials
Stock concentrations:
Protocol
Competence:
