Team:TU Darmstadt/protocols/Chemically competent cells

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<h2><font size="6" color="#F0F8FF" face="Arial regular"> Chemically competent cells </font></h2>
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<B> Short explanation<br></B>
The transformation of E. coli with plasmid DNA via heatshock transformation requires chemically competent cells.<br>
<br>
<B> Equipment <br></B>
-80°C freezer<br>
Incubation shaker<br>
Centrifuge (cooling cababilities required!)<br>
photometer<br>
Ice water bath<br>
<br>
<B> Chemicals & consumables <br></B>
Ice and/or liquid nitrogen<br>
Falcon tubes<br>
Eppis<br>
dYT Medium (50 ml p.c.)<br>
ice cold 100mM CaCl2<br>
Glycerin<br>
<br>
<B> Procedure<br></B>
1. Inoculate 2 mL of LB-Media with an E. coli colony and incubate at 37 °C overnight<br>
2. Inoculate 200 mL LB with the preculture <br>
3. Incubate at 37°C and 150 rpm until an OD600 of 0.4-0.6 is reached<br>
4. Incubate cells on ice for 15 min<br>
5. Centrifuge the culture at 4°C and 3000 x g for 10 min (the following steps are carried out on ice)<br>
6. Resuspend cell pellet in 10mL ice cold 100 mM CaCl2 (Do not vortex!)<br>
7. Incubate on ice for 1 hour<br>
8. Centrifuge the culture at 4°C and 3000 x g for 10 min<br>
9. Resuspend cell pellet in 10mL ice cold 100 mM CaCl2<br>
10. Incubate on ice for 1 hour<br>
11. Centrifuge the culture at 4°C and 3000 x g for 5 min<br>
12. Resuspend cell pellet in 2mL ice cold 100 mM CaCl2 and 15 % (v/v) glycerine<br>
13. Incubate on ice for 30 min<br>
14. Aliquot the cells à 100µL<br>
15. Store at -80°C<br>
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