how to make sigle-stranded DNA of pET

Karl Fischer kfischer at gpu.srv.ualberta.ca
Sat Nov 26 10:32:52 EST 1994


In article <Pine.SOL.3.91.941123113627.19327A-100000 at dale>, Yee-Yung
Charng <szcharng at peseta.ucdavis.edu> wrote:

> Hi, I wonder if anybody can give me a clue to make ssDNA of pET-based
> plasmid with good yield.  We have tried Novablue and XL1-blue as host 
> cells and USC-M13 as a helper phage without success. I was told to use 
> Promega's R408 helper phage instead.  Any input ?  Thanks in advance.
> 
> Yee-yung Charng
> yccharng at ucdavis.edu  

I don't use the strains of E.coli you mentioned but what I find works for
generating ssDNA for Promega's pSelect system is JM103 grown  overnight in
M9+casamino acids+vitamin B1 and your antibiotic for plasmid maintenance.
The next day inoculate with M13K07 (stock:10^8 - 10^10/ml), let the phage
adsorb for 1 hour at 37°C in a waterbath then use that culture to
inoculate a rich broth containing kanamycin (50 µg/ml final concentration)
and your original selective antibiotic. Incubate in a shaker (250 rpm)
overnight; good aeration is a necessity for good yield so we use
fluted-bottom flasks for this. The next day take 1.5 ml, spin to pellet
the cells, transfer 1 ml of sup to a fresh tube and precipitate with
PEG/NaCl. If you don't see a prominent pellet at this point then start
over ;-(



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