Difference between revisions of "Team:Ionis Paris/Protocol 5"
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<h4 class="sidebar_Hd">Sources</h4> | <h4 class="sidebar_Hd">Sources</h4> | ||
| − | <p><li><a href="http://www.med.nyu.edu/medicine/labs/blaserlab/Protocols/E-coli_competent_cells_protocol_&_transformation.pdf" | + | <p><li><a href="http://www.med.nyu.edu/medicine/labs/blaserlab/Protocols/E-coli_competent_cells_protocol_&_transformation.pdf"><font color="DeepPink">NYU Protocol for competent cell preparation</font></a></li></p> |
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| − | + | <p><li><a href="http://www.sb.fsu.edu/~mestroupe/protocols/CalciumChloride.pdf"><font color="DeepPink">FSU Protocol for competent cell preparation</font></a></li></p> | |
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Revision as of 15:05, 18 October 2016
Inoculate 100µL of cells in 100mL LB medium
Put 1.11g of CaCl2 in 100mL of sterile H2O to obtain a final concentration of 0.1M of CaCl2 (Molar mass of Cacl2: 110,98g/mol)
Inoculate 3mL of the overnight culture cell from in 90mL of LB. Shake at 37°C until the DO is around 0,6
Protocol 5 : Preparation of competent cells
Aim: Prepare competent bacteria for subsequent transformation
Inoculation
Let the culture grow O/N at 37°C
CaCl2 preparation
Competency
NB : all the following steps must be perform in ice
Transfer cells into sterile ice-cold Falcon tubes
Incubate on ice for 30 min
Spin cells at 4000 rpm for 10 min at 6°C
Discard supernatant and try to drain all remaining media
Gently resuspend in 10mL cold 0,1M CaCl2
Incubate on ice 20 min
Centrifuge 10 min at 4000 rpm at 4°C
Discard supernatant and vortex, gently resuspend on 5mL cold 0,1M CaCl2/15% Glycerol
Transfer in 2mL cryotubes
Store at -80°C for subsequent transformation
