Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

An unusual "buffer" in a protocol for making chemically competent E.coli

E.coli competent cells

  • Please log in to reply
3 replies to this topic

#1 Cistron

Cistron

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 06 September 2017 - 12:02 AM

My first post here - hello!

 

I've got given the following protocol for making competent cells (see below). It has been established in a lab in France for a long time, but even intensive searching has only thrown up some limited information about it.

 

What strikes me particularly odd is the treatment buffer ('FB' I assume stands for freezing buffer). It's not much of a buffer with KAc at a pH at 7.5 and then set to 6.2, as it's far off acetate's pKa. 

 

I know historically making competent cells was an arcane art and researchers like Hanahan would only give out cell aliquots and not divulge their painstakingly developed procedures.

 

Is this 'buffer' one of these odd quirks? Has anyone come across this before, maybe even this protocol?

 

Many thanks!

PREPARATION OF COMPETENT CELLS (FB METHOD)
 
 
• set up an overnight culture of TOP10F' or other E. coli strain in SOB (or LB) and let grow O/N with shaking
 
• inoculate 100 ml of SOB with 1 ml of the saturated O/N culture
 
• grow until OD550 = 0.45 to 0.55  (getting the OD right is important as it affects the transformation efficiency**)
 
• transfer culture to 2-50 ml Falcon tubes and chill on ice for 10'
 
• centrifuge 10' at 2500 rpm at 4°C
 
• resuspend each 50 ml cell pellet in 15 ml FB buffer
 
• let sit on ice for 10'
 
• centrifuge 10' at 5000 rpm at 4°C
 
• resuspend each pellet in 4 ml FB buffer with gentle pipetting
 
• aliquot cells, freeze in liquid nitrogen and store at -80°C
 
______________________________________________________________________
 
SOB   2% tryptone
                        0.5% yeast extract
                        10 mM NaCl
                        2.5 mM KCl
                        10 mM MgCl2**
                        10 mM MgSO4**
 
                        **  add after autoclaving
 
 
FB                   100 mM KCl
                        50 mM CaCl2
                        10% glycerol
                        10 mM KAc pH 7.5
 
                        adjust the pH of the solution to 6.2 with the addition of HCl
 
                        autoclave        
 

Getting the OD right…….
 
Ln[Y/start]/K = X
 
Where              Y = population size (cell number, OD etc.)
                        X = time (typically in minutes)
                        K = slope = rate constant; doubling time = 0.693/K
 
Have determined K experimentally twice for TOP10F’ (K=0.0183 – single determination; K=0.01974 – growth curve performed on 02 May 06)


#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 6,387 posts
512
Excellent

Posted 06 September 2017 - 06:23 AM

This is not an arcane method - it's actually a variant on the method used by the Registry of Standard Biological Parts, it works very well for preparing high competency chemically competent cells.

 

The full method with some info and links is here: http://www.openwetwa...competent_cells



#3 Cistron

Cistron

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 06 September 2017 - 07:49 AM

This is not an arcane method - it's actually a variant on the method used by the Registry of Standard Biological Parts, it works very well for preparing high competency chemically competent cells.

 

The full method with some info and links is here: http://www.openwetwa...competent_cells

Thanks bob! I don't think I'd have ever found this connection.

 

The buffers seem somewhat similar. I see they are also using acetate at quite a high pH.



#4 labtastic

labtastic

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 201 posts
20
Excellent

Posted 07 September 2017 - 07:58 AM

The only odd thing to me is autoclaving the FB buffer. Typically competent cell buffers are filter sterilized, especially if they have manganese or Good's buffers in them. But since this buffer has neither maybe it is OK. Though I still wouldn't because autoclaves, though sterile, are nasty dirty and I wouldn't want to potentially compromise a buffer for such a sensitive process like making chemically competent cells.

 

I can confirm the CCMB80 protocol does work very well. Though I like the rubidium chloride method personally. It takes a bit more time to make the buffers, and rubidium chloride isn't super cheap, but since switching over to that method transformations in our lab have been particularly robust. Students from other labs steal our competent cells quite frequently...I have to beat them off with a stick when I catch these thieves. wink.png


Edited by labtastic, 07 September 2017 - 08:00 AM.






Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.