Team:TU Darmstadt/protocols/Chemically competent cells

(Difference between revisions)

Revision as of 23:45, 4 October 2013

Materials

Equipment

Chemicals & consumables

Procedure

The transformation of E. coli with plasmid DNA via heatshock transformation requires chemically competent cells.

Inoculate 2 mL of LB-Media with an E. coli colony and incubate at 37 °C overnight.
Inoculate 200 mL LB with the preculture.
Incubate at 37°C and 150 rpm until an OD600 of 0.4-0.6 is reached.
Incubate cells on ice for 15 min.
Centrifuge the culture at 4°C and 3000 x g for 10 min (the following steps are carried out on ice).
Resuspend cell pellet in 10mL ice cold 100 mM CaCl₂ (Do not vortex!).
Incubate on ice for 1 hour.
Centrifuge the culture at 4°C and 3000 x g for 10 min.
Resuspend cell pellet in 10mL ice cold 100 mM CaCl₂.
Incubate on ice for 1 hour.
Centrifuge the culture at 4°C and 3000 x g for 5 min.
Resuspend cell pellet in 2mL ice cold 100 mM CaCl₂ and 15 % (v/v) glycerine.
Incubate on ice for 30 min.
Aliquot the cells à 100µ.
Store at -80°C.

Mixtures

CaCl2-Solution

Cryo solution

Mandel, M. and Higa, A.: Calcium-dependent bacteriophage DNA infection. J Mol Biol, 1970, 53, 159-162

@@ Line 268: / Line 268: @@
 <li class=list1>- Ice and/or liquid nitrogen</li>
 <li class=list1>- Falcon tubes</li>
-<li class=list1>- dYT Medium (50 ml p.c.)</li>
+<li class=list1>- <a href="https://2013.igem.org/Team:TU_Darmstadt/materials/Media"> dYT Medium</a>(50 ml p.c.) </li>
 <li class=list1>- ice cold 100mM CaCl<sub>2</sub></li>
 <li class=list1>- Glycerin</li>