competence in e. coli

[Jonathan Nesser]

I posting this as another question in the form of "I must be crazy, please correct me." My understanding of induced competence in e. coli is that it involves creating a new culture in LB medium, soaking in a CaCl solution (other components can be added for the perfectionist), cooling and then heat shocking, as well as being very gentle with the cells (no vortexing, etc). I know that many companies sell e. coli cultures that have been deemed competent, but is there any reason why using the above procedure on new cultures from other sources which are incompetent would not yield competent cells?

 

My problem with the standard lab method of simply ordering competent cells is (besides the obvious expense factor), that I don't have any means of storing the competent cells I receive at -80C, which I'm told destroys the company's competence work anyways. Also, I've discovered that I can obtain cultures for the Coli Genetic Stock Center at Yale for substantially less (less than half that which New England Biolabs charges, and the price for extra strains in the same order increases te savings exponentially). It seems like I must be missing something somewhere, because it doesn't make sense that large labs wouldn't induce competence themselves (unless it's just a time saving and quality assurance factor).

 

Also note that I have read that most "home" induced competence jobs yield a lower transformation rate, but figure that with selection and time to grow cultures I should be able to yield a decent number of transformed cells in the end, and in the case of everything I will be doing in the next half year, there is no product I'm looking to yield (unless I get into cubcloning), just the confirmation that my transformed plasmid works.

 

Someone please set me straight, and as always, I apologize for the simple question, and deeply appreciate the time spent answering my questions.

 

Jonathan Nesser

jonatha...@gmail.com

diybioandneurosci.blogspot.com

[Jacob Shiach]

The CaCl protocol you mentioned IS the protocol for making competent cells. So it will yield competent cells.

-

Jacob Shiach

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[Cathal Garvey]

And, leaving aside that CaCl *will* give you loads of competent cells,
there's an even easier method out there that makes the very idea of
buying competent cells seem preposterous. I've posted on it before in
Mega's previous thread on competence. It's much easier than the CaCl
method, requiring no real preparation at all; take fresh cells, add
buffer, add DNA, chill on ice, then select. That's it.

The *only* valid reason (I think!) to buy expensive competent cells
rather than just hammering out your own on-demand is if you are doing
"Libraries": Transformation procedures where every transformed bacteria
has a different combination of DNA, and you want to collect as many
transformed bacteria as you possibly can. For these procedures,
transformation efficiency is critical, but for all others it's not that
important beyond a certain threshold.

And, because the CaCl and PEG/MgSO4 methods yield plenty of
transformants, that threshold is firmly beaten into the soil as far as
I'm concerned.

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[Nathan McCorkle]

On Fri, Feb 3, 2012 at 1:22 PM, Jacob Shiach <king...@gmail.com> wrote:
> The CaCl protocol you mentioned IS the protocol for making competent cells.
> So it will yield competent cells.
>

That's not totally true, CaCl2 is ONE method for making competent
E.coli cells, there are others, for instance those outlined in
"molecular cloning a laboratory manual" by Sambrook... competency
isn't an exact state either (as you said, CaCl2 transformation
protocol uses log-phase cells, but they're not really prepared for
maximum competency... some might not call them competent at all) .
Some methods prepare E.coli with special buffers and minimal medias
for CaCl2 TRANSFORMATION, some prepare cells for electroporation
transformation, or the PEG MgSO4 method that Cathal has told us about.

The third message from me in this post has all the info I compiled for
a class I taught where we used CaCl2 transformation:
http://groups.google.com/group/diybio/browse_thread/thread/fa9fd3c4df71d8d0?pli=1

--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

[Laureana Stelmastchuk]

Well, it wont answer your main questions, but could give you some great ideas...
Have you ever tried electroporation? (http://en.wikipedia.org/wiki/Electroporation#Electroporators).
I think electrocompetent cells are easier to prepare than the chemical ones, and it shows better transformaton rates. I have used both E.coli DH10B and DH5α.
However, if you don't have an electroporator... I don't know, but would be cool to try to make one.

2012/2/3 Jonathan Nesser <jonatha...@gmail.com>

 

Jonathan Nesser

jonatha...@gmail.com

diybioandneurosci.blogspot.com

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Laureana Stelmastchuk Benassi Fontolan
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ICB II - USP

[Mega]

That'll be one of my next projects... :) An electroporator....

Using an piezoelectric lighter.... Has anyone a foto of the circuit?
You have to push the piezoelectric botton with your hands very often,
or is there a circuit that transforms the voltage??

On 3 Feb., 19:55, Laureana Stelmastchuk <laure.stelmastc...@gmail.com>
wrote:

> Well, it wont answer your main questions, but could give you some great
> ideas...
> Have you ever tried electroporation? (http://en.wikipedia.org/wiki/Electroporation#Electroporators).
> I think electrocompetent cells are easier to prepare than the chemical

> ones, and it shows better transformaton rates. I have used both *E.coli *DH10B

> and DH5α.
> However, if you don't have an electroporator... I don't know, but would be
> cool to try to make one.
>

> 2012/2/3 Jonathan Nesser <jonathan.nes...@gmail.com>

> > jonathan.nes...@gmail.com

[Nathan McCorkle]

one or two sparks should be sufficient as long as the volts/cm is right.

--

[Nick Beck]

On the subject of electroporation, are there any open source designs
around? Do we have a list of vendors of used science equipment that
sell reliable goods?

Sorry if this is said elsewhere!

> > For more options, visit this group athttp://groups.google.com/group/diybio?hl=en.

[Mega]

You don't need any resistors or capacitors or ... anything?

Just an ignitor, electrodes and a cuvette??????

If so, Awsome!!!

> > For more options, visit this group athttp://groups.google.com/group/diybio?hl=en.