E. coli DH5 alpha culture problem

Kyle Legate via methods%40net.bio.net (by legatekBLAH from hotmail.com)
Sat Feb 28 03:09:52 EST 2009


peter wrote:

> Well, you have to seed pre-culture single colony in say 5-10 ml LB
> +Abt, and then innoculate the 500 flask.
> my2c

I agree, the seeding culture was probably too small and not ready to add 
to the 500 mL culture, resulting in an unacceptably long lag phase. It 
should be 5-10 mL as Peter said and you should not be able to see 
through it when you add to the larger flask. Some people spin down the 
starter culture and resuspend in fresh LB-Amp to remove the secreted 
beta-lactamase but personally I don't do this for plasmid amplification. 
The long lag phase you experienced due to a dilute seed culture 
containing beta-lac may have depleted the antibiotic, resulting in some 
of the bacteria losing the plasmid, resulting in low yield.

For a high yield of plasmid you can grow DH5a in Superbroth: 32g 
tryptone, 20g yeast extract, 5g NaCl, 1.25mL 4M NaOH per litre. I 
routinely get >1 mg of DNA per 100 mL of culture with this recipe.


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