plasmid cDNA library amplification

TJ Murphy medtjm at bimcore.emory.edu
Sun Mar 31 03:21:06 EST 1996


CartonKJ wrote:
> 
> I am trying to make a cDNA library in the pSPORT vector (gibco).  So far
> everything has gone well, however, the protocol I am following ends
> without describing the amplification of the library.  I was wondering if
> anyone has a protocol they have used to successfully amplify a plasmid
> library and if you would please share it with me.
> 
> Thanks very much,
> Jill

You have lot's of choices, and what to do depends upon your goals.  I 
personally think there is less effort in making a new plasmid library next 
time you need it than in going to the trouble of amplifying a plasmid library 
correctly.  If it's a tissue you hope to go back to time and again for 
clones, you'd be better off having made a phage library.

I prefer to make the library (usually by electroporation), and after 
recovering the bacteria, remove a small amount to titer and save the rest 
unamplified as 10 or so frozen bacterial aliquots.  Over the next few days, I 
get the titer and quality (% recombinants, average insert size and range) and 
then decide what to do.  

If it's a great libary, (>1 million clones, >90% recombinant, average insert 
of > 2 kb), I might amplify half of it over the next few weeks for the sake 
of posterity, but it's a lot of work, and probably more so than simply making 
a new library next time you need one (the second always goes easier than the 
first).

If it's a good but not great libary, I won't have screwed it up by amplifying 
it without knowing anything about it.  For instance, a good library might 
have >80% recombinants with an average insert size of ~ 2kb, but maybe less 
than 500,000 recombinants.  I might not want to screen it if I had amplified 
all of it first, but I'd likely still screen something like this if 
unamplified.

The screening itself involves an amplification in order to get enough DNA or 
colonies to work with.  So if you amplify after making it, it will be 
amplified again while you screen it.  It's a risk that you'll select against 
your target by doing this twice.

Whether or not it's the first or second, it's best to amplify on agar rather 
than in liquid culture (IMHO). In the latter, it is much more likely that 
clones will be under-represented, as faster growing clones tend to overwhelm 
the liquid cultures and if your target is not one of these...

To do this, plate out pools of anywhere between 500-25,000 clones on 10 or 
150 mm agar plates (use the latter for pool sizes >1000 clones). Simply 
scrape the bacteria from the plates after overnight colony formation and save 
bacterial aliquots as 7-10% DMSO or 10% glycerol stocks.  Prepare DNA 
aliquots from the remainder.  

You can screen the DNA by transfection into cells, by running off transcripts 
in vitro, or by making master southern blots.  Simply return to the bacterial 
stocks of the pools and sib select out a positive clone from one of these.

Another choice is to save half of your library as unamplified (and amplify as 
you go) and amplify the other half just after creating it.   Problem is you 
won't know until you've got and analyzed your titer how good the library is, 
and it's really only worth the risk (IMHO) of doing this if your library has 
a few million EXCELLENT recombinants. About the only logical/efficient way of 
doing a blind amplification just after creation is in liquid culture, which 
I've discussed above as being suboptimal.



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