Competent cells

Daniel Prieto via methods%40net.bio.net (by dprieto from fcien.edu.uy)
Mon Jul 26 14:15:44 EST 2010


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"<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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