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.
On 03/02/12 18:02, Jonathan Nesser wrote:
> 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
> jonathan.nesser@gmail.com
> diybioandneurosci.blogspot.com
>
--
www.indiebiotech.com
twitter.com/onetruecathal
joindiaspora.com/u/cathalgarvey
PGP Public Key: http://bit.ly/CathalGKey
--
You received this message because you are subscribed to the Google Groups "DIYbio" group.
To post to this group, send email to diybio@googlegroups.com.
To unsubscribe from this group, send email to diybio+unsubscribe@googlegroups.com.
For more options, visit this group at http://groups.google.com/group/diybio?hl=en.
0 comments:
Post a Comment