Team:TU Darmstadt/Protocols/Chemically competent cells

From 2012.igem.org

(Difference between revisions)
(→Chemically competent cells)
(→Procedure)
Line 55: Line 55:
* ice cold 100mM CaCl<sub>2</sub>  
* ice cold 100mM CaCl<sub>2</sub>  
=== Procedure ===
=== Procedure ===
-
# 2mL preculture ''E.coli'' in DYT-medium
+
# Inoculate 2 mL of LB-Media with an E. coli colony and incubate at 37 °C overnight
-
# incubate preculture overnight at 37°c
+
# Inoculate 200 mL LB with the preculture
-
# inoculate 200 mL with the preculture
+
# Incubate at 37°C and 150 rpm until an OD<sub>600</sub> of 0.4-0.6 is reached
-
# incubate at 37°C and 150 rpm till an OD<sub>600</sub> of 0.4 - 0.6 is reached
+
# Incubate cells on ice for 15 min
-
# 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)
-
# centrifuge the culture at 4°C and 3000 x g for 10 min (the following steps are done on ice)
+
# Resuspend cell pellet in 10mL ice cold 100 mM CaCl<sub>2</sub> (Do not vortex!)
-
# resuspend cell pellet in 10mL ice cold 100 mM CaCl<sub>2</sub>
+
# Incubate on ice for 1 hour
-
# incubate on ice for 1 hour
+
# Centrifuge the culture at 4°C and 3000 x g for 10 min
-
# centrifuge the culture at 4°C and 3000 x g for 10 min
+
# Resuspend cell pellet in 10mL ice cold 100 mM CaCl<sub>2</sub>
-
# resuspend cell pellet in 10mL ice cold 100 mM CaCl<sub>2</sub>
+
# Incubate on ice for 1 hour
-
# incubate on ice for 1 hour
+
# Centrifuge the culture at 4°C and 3000 x g for 5 min
-
# centrifuge the culture at 4°C and 3000 x g for 5 min
+
# Resuspend cell pellet in 2mL ice cold 100 mM CaCl<sub>2</sub> and 15 % (v/v) glycerine
-
# resuspend cell pellet in 2mL ice cold 100 mM CaCl<sub>2</sub> and 15 % (v/v) glycerine
+
# Incubate on ice for 30 min
-
# incubate on ice for 30 min
+
# Aliquot the cells à 100µL
-
# aliquot the cells à 100µL
+
# Store at -80°C
-
# store at -80°C
+
 
 +
=== Solutions ===
 +
* CaCl<sub>2</sub>
 +
**5.55 g CaCl<sub>2</sub>
 +
**Add di H<sub>2</sub>O to 1 L
 +
**Sterilize by autoclaving
 +
 
 +
*Cryo solution
 +
** 0.278 g CaCl<sub>2</sub>
 +
** 10 ml glycerin
 +
** Add di H<sub>2</sub>O to 50 ml
 +
** Sterilize by autoclave
 +
 
== References ==
== References ==
* Mandel, M. and Higa, A.: Calcium-dependent bacteriophage DNA infection. J Mol Biol, 1970, 53, 159-162
* Mandel, M. and Higa, A.: Calcium-dependent bacteriophage DNA infection. J Mol Biol, 1970, 53, 159-162

Revision as of 19:04, 16 September 2012

Contents

Chemically competent cells

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

Procedure

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

Solutions

  • CaCl2
    • 5.55 g CaCl2
    • Add di H2O to 1 L
    • Sterilize by autoclaving
  • Cryo solution
    • 0.278 g CaCl2
    • 10 ml glycerin
    • Add di H2O to 50 ml
    • Sterilize by autoclave

References

  • Mandel, M. and Higa, A.: Calcium-dependent bacteriophage DNA infection. J Mol Biol, 1970, 53, 159-162
Retrieved from "http://2012.igem.org/Team:TU_Darmstadt/Protocols/Chemically_competent_cells"