This is strange.
I've used the pET-Duet and pACYCDuet vectors extensively and the
others once or twice and I've never seen behavior like that.
The pET-Duet has the same replication origin as "normal" pET vectors
and has never presented me with any more difficulty than they do.
I typically use the pACYCDuet (in order to coexpress with a protein
already cloned into a pET vector) and it has also behaved normally.
They are low copy number, but a standard miniprep (performed on a cell
pellet from 10 mL culture) gives me ~150 ng/µL in 50 µL, which is
plenty to work with for a long time.
So, I would qualify my experience with them as "successful".
Are you seeing the decrease in DNA yield as your plates age, or are
these from fresh transformations?
What strain do you use for plasmid propagation?
What medium do you use for growth to prepare plasmids?
How do you perform your plasmid preps?
Best of luck,
Cynthia
On Oct 29, 2009, at 9:11 AM, Silvia Onesti wrote:
Dear all,
We are attempting to clone into pETDuet, pACYCDuet, pCola1Duet and
pCDFDuet and we are encountering numerous difficulties.
We are aware that most of them are low copy number, but even taking
this into account, we find it puzzling that whereas the first preps
of the vectors gave low, but significant amount of DNA, as time goes
by the quantities we get are constantly decreasing. And any
subsequent step (ligation, etc.) is a real challenge.
Does anyone have any experience (succesful or unsuccessful) with
these vectors?
Many thanks,
Silvia
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Silvia Onesti
Sincrotrone Trieste S.C.p.A.
SS14 - km 163,5 in AREA Science Park
34149 Basovizza, Trieste ITALY
Tel: +39 040 3758451
Mob: +39 366 6878001
Email: silvia.one...@elettra.trieste.it
http://www.elettra.trieste.it/PEOPLE/index.php?n=SilviaOnesti.HomePage
http://www.sissa.it/sbp/web_2008/C_Structural_Biology.html
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__________________________________
Cynthia Kinsland, Ph. D.
Protein Production and Characterization Facility
Cornell University
B78 ST Olin Lab Bldg.
Ithaca, NY 14853-2703
Tel: (607) 255-8844
E-mail: cl...@cornell.edu
