plasmid cDNA library amplification

john brennand john.brennand at gbapr.zeneca.com
Mon Apr 1 04:20:10 EST 1996


Jill

I'm not convinced you need to re-amplify a plasmid library beyonmd the 
original plating out.

After ligation of cDNA to vector, test transform to determine the 
potential library size. Then, scale up to generate 1 million colonies 
split between 20 plates.  Scrape off in 5-10 ml L-broth/plate and remove 
1-2 ml for 20 plasmid DNA preps & store the rest in the 20 aliquots 8% 
DMSO at -80. You will get 10's of ug of DNA.

You now have DNA's from 20 aliquots of your library each corresponding to 
50,000 clones and you can titre the frozen aliquots or pool them if 
required.

If you want to ensure that the library isn't skewed by "larger" colonies, 
plating into top agar is reported to standardise the colony size - 
although we have never needed to do this.

John





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