Team:TU Darmstadt/protocols/Chemically competent cells

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Revision as of 19:02, 4 October 2013

Chemically competent cells

Materials

Equipment

- -80°C freezer
- Incubation shaker
- Centrifuge (cooling cababilities required!)
- photometer
- Ice water bath

Chemicals & consumables

- Ice and/or liquid nitrogen
- Falcon tubes
- dYT Medium (50 ml p.c.)
- ice cold 100mM CaCl₂
- Glycerin

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

- 5.55 g CaCl₂
- add ddH₂O to 1 L
- sterilize by autoclaving

Cryo solution

- 0.278 g CaCl₂
- 10 ml glycerin
- Add ddH₂O to 50 ml
- Sterilize by autoclave

References

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

@@ Line 210: / Line 210: @@
 <li>Incubate cells on ice for 15 min.</li>
 <li>Centrifuge the culture at 4°C and 3000 x g for 10 min (the following steps are carried out on ice).</li>
-<li>Resuspend cell pellet in 10mL ice cold 100 mM CaCl2 (Do not vortex!).</li>
+<li>Resuspend cell pellet in 10mL ice cold 100 mM CaCl<sub>2</sub> (Do not vortex!).</li>
 <li>Incubate on ice for 1 hour.</li>
 <li>Centrifuge the culture at 4°C and 3000 x g for 10 min.</li>
-<li>Resuspend cell pellet in 10mL ice cold 100 mM CaCl2.</li>
+<li>Resuspend cell pellet in 10mL ice cold 100 mM CaCl<sub>2</sub>.</li>
 <li>Incubate on ice for 1 hour.</li>
 <li>Centrifuge the culture at 4°C and 3000 x g for 5 min.</li>
-<li>Resuspend cell pellet in 2mL ice cold 100 mM CaCl2 and 15 % (v/v) glycerine.</li>
+<li>Resuspend cell pellet in 2mL ice cold 100 mM CaCl<sub>2</sub> and 15 % (v/v) glycerine.</li>
 <li>Incubate on ice for 30 min.</li>
 <li>Aliquot the cells à 100µ.</li>
@@ Line 230: / Line 230: @@
 <div align="left" style="margin-left:60px; margin-right:50px">
 <ul>
-<li class=list1>- 5.55 g CaCl2</li>
+<li class=list1>- 5.55 g CaCl<sub>2</sub></li>
-<li class=list1>- add ddH2O to 1 L</li>
+<li class=list1>- add ddH<sub>2</sub>O to 1 L</li>
 <li class=list1>- sterilize by autoclaving</li>
 </ul>
@@ Line 242: / Line 242: @@
 <div align="left" style="margin-left:60px; margin-right:50px">
 <ul>
-<li class=list1>- 0.278 g CaCl2</li>
+<li class=list1>- 0.278 g CaCl<sub>2</sub></li>
 <li class=list1>- 10 ml glycerin</li>
-<li class=list1>- Add ddH2O to 50 ml</li>
+<li class=list1>- Add ddH<sub>2</sub>O to 50 ml</li>
 <li class=list1>- Sterilize by autoclave</li>
 </ul>