. .
.
Preparation of Competent Cell (Calcium Chloride Treatment)
.
.

 

Materials Required:-

 

  •  LB broth
  •  Culture plates
  •  Ice cold CaCl2.2H2O  (1  M)
  •  Ice cold MgCl2 CaCl2  solution  
  •  Shaking incubator
  •  Vortex mixer
  •  Centrifuge
  •  Water  bath
  •  Inoculation loop
  •  Microfuge tubes
  •  Polypropylene  tubes
  •  Micro pipettes and tips

 

Procedure:-

 

  1. Pick a single bacterial colony  from a culture plate which is incubated for 16-20 hours at 37°C. Transfer the colony into 100 ml LB broth a 1-liter flask. Incubate the culture for 3 hours at 37°C with vigorous agitation, monitoring the growth of the culture. As a guideline, 1 OD600 of a culture of E. coli strain DH 5 alpha contains approx. 109 bacteria/ml.
     
  2. Transfer the bacterial cells to sterile, disposable, ice-cold 50ml polypropylene tubes. Cool the cultures to 0°C by storing them on ice for 10 minutes.
     
  3. Recover the cells by centrifugation at 2700g  at 4°C for 10 minutes .
     
  4. Decant the medium from the cell pellets. Stand the tubes in an inverted position on a pad of paper towels for 1 minute to allow the last traces of media to drain away.
     
  5. Resuspend each pellet by swirling or gentle vortexing in 30 ml of ice-cold MgCl2-CaCl2 solution.
     
  6. Recover the cells by centrifugation at 2700g  at 4°C for 10 minutes .
     
  7. Decant the medium from the cell pellets. Stand the tubes in an inverted position on a pad of paper towels for 1 minute to allow the last traces of media to drain away.
     
  8. Resuspend the pellet in  2 ml of ice-cold 0.1 M CaCl2 (or TFB) by gentle vortexing for each 50ml of original culture. Standard TFB may be used instead of CaCl2 for most strains of E. coli.
     
  9. At this point, either use the cells directly for transformation or dispense into aliquots and freeze at -70°C.

 

 

 

Help:

 

                    Unfortunately, this Virtual Lab requires Adobe Flash player.  Please see additional information if this does not work on your computer


• For Google Chrome - https://support.google.com/chrome/answer/6258784?co=GENIE.Platform%3DDesktop&hl=en

• For Microsoft Edge - https://support.microsoft.com/en-in/help/4532571/microsoft-edge-turn-on-flash 

• For Mozilla Firefox - https://support.mozilla.org/en-US/kb/install-flash-plugin-view-videos-animations-games

 

On GNU/Linux machines, please see appropriate online help. For example, 

 

• on Ubuntu - https://help.ubuntu.com/stable/ubuntu-help/net-install-flash.html.en 

• on Fedora - https://docs.fedoraproject.org/en-US/quick-docs/using-adobe-flash/

 

 

 

Cite this Simulator:

.....
..... .....

Copyright @ 2020 Under the NME ICT initiative of MHRD

 Powered by AmritaVirtual Lab Collaborative Platform [ Ver 00.13. ]