"
Team:TU Darmstadt/Protocols/Electrocompetent cells
From 2012.igem.org
(Difference between revisions)
(Created page with "<html> <link rel="stylesheet" href="https://2012.igem.org/wiki/index.php?title=Team:TU_Darmstadt/css&action=raw&ctype=text/css" type="text/css" /> <div id="TUD"> <div id="...") |
|||
| (One intermediate revision not shown) | |||
| Line 27: | Line 27: | ||
<li><a href="/Team:TU_Darmstadt/Safety" title="Safety">Safety</a></li> | <li><a href="/Team:TU_Darmstadt/Safety" title="Safety">Safety</a></li> | ||
<li><a href="/Team:TU_Darmstadt/Downloads" title="Downloads">Downloads</a></li></ul></li> | <li><a href="/Team:TU_Darmstadt/Downloads" title="Downloads">Downloads</a></li></ul></li> | ||
| - | <li><a href="/Team:TU_Darmstadt/Human_Practice" title="Human Practice">Human Practice</a | + | <li><a href="/Team:TU_Darmstadt/Human_Practice" title="Human Practice">Human Practice</a></li> |
| - | + | ||
| - | + | ||
| - | + | ||
<li><a href="/Team:TU_Darmstadt/Sponsors" title="Sponsors">Sponsors</a><ul> | <li><a href="/Team:TU_Darmstadt/Sponsors" title="Sponsors">Sponsors</a><ul> | ||
<li><a href="/Team:TU_Darmstadt/Sponsors" title="Sponsors">Overview</a></li> | <li><a href="/Team:TU_Darmstadt/Sponsors" title="Sponsors">Overview</a></li> | ||
| Line 65: | Line 62: | ||
=== References === | === References === | ||
| + | * Praktikumsskript Biotechnologie 2012 TU Darmstadt | ||
Latest revision as of 21:13, 26 September 2012
Contents |
Electrocompetent cells
The transformation of E. coli with plasmid DNA via electroporation according to Dower et al. requires electrocompetent cells.
Materials
Equipment
- -80°C freezer
- Incubation shaker
- Centrifuge (cooling cababilities required!)
- OD meter
- Ice water bath
Chemicals & consumables
- Ice and/or liquid nitrogen
- Falcon tubes
- dYT Medium (50 ml p.c.)
- ice cold ddH2O (95+ ml p.c.)
Procedure
- Cultivate the cells in 50 ml dYT with a starting OD600 of 0.1 at 37°C and 180 U/min
- At an OD600 of 0.5 transfer the cells to a sterile Falcon tube
- harvesting of the cells is performed with centrifugation at 4000 U/min 4°C
- Wash the cells with an decreasing volume of 50/30/15 ml (discard the supernatant)
- use some of the supernatant in the last centrifugation step to resuspend the cells
Notes
- Important: Cooling must be maintained at all times.
References
- Praktikumsskript Biotechnologie 2012 TU Darmstadt
