Amplyfing cDNA libraries

Clive Tregaskes Clive.tregaskes at bbsrc.ac.uk
Fri Dec 13 12:26:31 EST 1996


iayork at panix.com (Ian A. York) wrote:

>In article <19961211231200.SAA09064 at ladder01.news.aol.com>,
>Robmit <robmit at aol.com> wrote:
>>
>>I have a plasmid cDNA library that I need to Amplify.  Is there any good
>>way of doing this other than plating out 100 150mm plates?  Can I grow it
>>up in liquid?

>I think that plates are generally accepted to be the best way of keeping
>the proportions of your inserts reasonably accurate - otherwise you end up
>with more of a problem with overgrowth of some inserts at the expense of
>others.

>I don't think you need anywhere near 100 plates, though, since you can
>plate 50,000 colonies + per plate without getting them too crowded; if you
>want around a million colonies, then 20 plates should do you.

>Ian
>-- 
>      Ian York   (iayork at panix.com)  <http://www.panix.com/~iayork/>
>      "-but as he was a York, I am rather inclined to suppose him a
>       very respectable Man." -Jane Austen, The History of England

I agree about growing them on plates as opposed to in solution.  To
save on work and get even growth we use NUNC bio assay dishes,
effectively as 20cm square petri dishes. To spread the bugs out evenly
we dilute them to 4-5ml and add approx. 10ml of autoclaved glass beads
3-4mm diameter. Just shake these all over the surface until the liquid
is absorbed, invert the plates and incubate. You may lose some
bacteria stuck to the beads, but the remainder are spread out fairly
evenly.
Clive Tregaskes
Institute for Animal Health
Compton
UK
clive.tregaskes at bbsrc.ac.uk




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