I agree that Inoue's protocol works great but it's kind of laborious if
you only want to revive a couple of 'normal' plasmids. There is a very
straightforward one by Chung et al. (PNAS 1989, 86:2172-2175) which I
can assure you it works great and you don't even have to prepare your
cells beforehand. Not very high efficiency - as Inoue's which I would
say it's for supercompetent cells - but very easy and efficient enough
if you don't have a very large plasmid. I wouldn't use it for cloning
but it's what I would use for routine amplification of a plasmid.
Hanahan's protocol (traditional CaCl2) also works beautifully but it
takes more time and I wouldn't store that cells for later use, let's say
1 week at 4ºC.
Good luck,
Daniel
El 25/07/10 08:56, Cathal Garvey escribió:
Thanks, that's really helpful to know! I may have to invest in some high
volume filters early on in that case.
All the best,
Cathal
---
Twitter: @onetruecathal
Sent from my beloved Android phone.
On 25 Jul 2010 03:23, "DK"<[email protected]> wrote:
In article<[email protected]>, Cathal Garvey
<[email protected]...
Simple calcium chloride cells are super easy to make but their
transformation efficiency tops at 10^6/ug (usually an order less).
There are many protocols that give higher efficiencies for
CaCl2 cells but they seem to be poorly reproducioble. The most robust
of all chemical protocols is Inoue method, whuch us great. Gives
around 10^8/ug in our hands (don't think we ever hit 10^9 stated in
the paper). Its only downside is that the frozen cells seem to slowly
lose transformation efficiency over time. And there are some
applications where higher efficiency with electroporation matters.
Google +Inoue +transfrmation +E.coli. Growing cells at low temp
is a critical part of Iniou method, so if you don't have refrigerated
chaker, wrap flasks in towels and spray water on them to keep
cells cool.
Has anyone got a straightforward protocol for chemically competent cells to
add? One which might ...
I can't be affirmative here but my sense is that it matters to an
extent to all conventional methods.
DK
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