Dear Silvia,
we have cloned genes in pETDuet, pACYCDuet and pCDFDuet without any
problems.
What we always do, with any plasmid, is transform bacteria freshly
before any miniprep or protein preparation - we do not believe in
stocking plasmids "on plate" or as glycerol stocks. Glycerol stocks
are for bacteria only - plasmids should be stored pure (in my
opinion). The reason is that bacteria are very "smart" in accumulating
mutations that lower expression, i.e. a mutant expressing less or no
protein will always outgrow the others.
Mark
Mark J. van Raaij
Dpto de Bioquímica, Facultad de Farmacia
Universidad de Santiago
15782 Santiago de Compostela
Spain
http://web.usc.es/~vanraaij/
researcherID: B-3678-2009
On 29 Oct 2009, at 14:11, 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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