Competent cells

Cathal Garvey via methods%40net.bio.net (by cathalgarvey from gmail.com)
Sun Jul 25 06:56:56 EST 2010


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" <dk from no.email.thankstospam.net> wrote:

In article <mailman.904.1280006602.25217.methods from net.bio.net>, Cathal Garvey
<cathalgarvey from gmail.com...
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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