ssDNA from pETs ?

Zophonias O. Jonsson zjons at vetbio.unizh.ch
Sun Jan 22 10:51:11 EST 1995


Dear colleagues!

  I have been trying to isolate dUMP containing ssDNA from pET23(+)
vectors, by growing them in the strain CJ236 and infecting with the helper
phage M13KO7.  In theory, at least, this should work since this pET bears
the F1 ori.  Unfortunately, however, In real life it doesn't work for me,
so If anyone hase produced pET derived ssDNA I would very much like his/her
advice.

Some facts:

1. My positive controls work.  I get a band with pBluescript although in
rather low yields.
2. The helper phage ssDNA band is visible, altough faint, so the cells have
probably not lost their F' episome.
2. The same batch of helper phage works very well with pBluescript in
XL1-Blue cells, using the same procedure.  (I have not tried to isolate pET
ssDNA from XL-Blue.)

Some questions:

1. Would you recommend a different helper phage or a different dut- /ung-
strain?
2. Do you have a fool-proof protocol for isolating ssDNA from low copy
number plasmids, such as the pETs?
3. Should I just forget it !!!?


Sincerely

Zophonias O. Jonsson
Institute of Veterinary Biochemistry
University of Zurich-Irchel
Switzerland



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