Thierry
We see the same thing...I always just "assumed" the additional shorter
band (if present) are/were denatured plasmid from the mini-prep that
cannot be cut by RE's.
We too only use XL 10-Gold cells because they accept very large
plasmids without need for electroporation. We have used SURE cells also
to limit re-arrangements but I find they don't transform as well as the
XL cells when it comes to vectors+insert where the sizes exceed 17 kbp.
SURE cells are decent for less than 3kbp inserts but should also be
grown at 30C or less to maintain integrity of the insert and plasmid
(especially if making libraries).
What we do to avoid re-arrangements entirely with XL cells is to grow
them at no more than 30C (often less...even room temp over the weekend)
and use half the concentration of AMP or less (20-25 ug/mL). Satellite
colonies have never been a problem but the plasmid and insert remain
stable...no unwanted deletions of homopolymers etc. (some homopolymers
involve over 100 repeats of the same trinucleotide) and polyprolines are
far more stable using this method as well. Of course it adds a day, but
in the end you usually get what you want cloned and without the headache
of errors.
We have also sequenced some of the pDM vectors across the act15
promoter and have found small errors (deletions or repeats) in the
AT-rich regions. They do not seem to affect expression but I expect they
were accurate when submitted to dictyBase and have undergone mutation in
the E. coli the Center uses, the AMP concentrations and the growth
temperatures.
My 2 cents.
Mike
On 2013-02-12 2:07, Thierry Soldati wrote:
> Dear Kristi,
>
> Yes, we do ! And I have discussed that often with Douwe and Robert
> Insall's people …
> In our hands, these vectors are excellent for must of their purposes
> … but they are big and unstable.
> We have to use XL10 Gold, which are usually reserved for genomic
> library cloning. We transform, pick a series of colonies and grow
> them
> in high Amp, at 30°C, overnight, and only for minipreps. We verify
> that the restriction patterns are OK, and we pool them. But for some
> clonings, we have had terrible rearrangements etc… and additional
> bands, most usually from shorter versions, appear if we attempt to
> grow them longer or in larger volumes.
> Hope this helps ?
> Cheers,
>
> Thierry
>
> On 11 Feb 2013, at 23:24, Kristi Miller <[log in to unmask] [1]>
> wrote:
>
>> Hello!
>>
>> I am working with the pDM310 vector. Has anyone designed primers
>> that they know to work well for sequencing? Also, when digesting the
>> constructs that I have made with the pDM310 vector with an insert I
>> sometimes see additional bands that do not make sense... Has anyone
>> else encountered this problem? Is there a chance that the pDM310
>> vector even after digestion becomes strangely supercoiled?
>>
>> Thanks!
>> Kristi
>>
>> Kristi E. Miller
>> Graduate Teaching Assistant
>> Biology Department
>> Dow Hall Room 246
>> Central Michigan University
>> Mount Pleasant, MI 48859
>
> --
> ================================================
> Prof. Thierry Soldati
> Department of Biochemistry
> University of Geneva
> 30 quai Ernest Ansermet, Sciences II
> CH-1211-Genève-4, Switzerland
>
> Tel: +41-22-379-6496
> Fax: +41-22-379-3499
> email: [log in to unmask] [2]
> http://cms.unige.ch/sciences/biochimie/-Thierry-Soldati-.html [3]
> ================================================
>
>
> Links:
> ------
> [1] mailto:[log in to unmask]
> [2] mailto:[log in to unmask]
> [3] http://cms.unige.ch/sciences/biochimie/-Thierry-Soldati-.html
--
----------------------------------
Michael A. Myre, PhD
Center for Human Genetic Research
Massachusetts General Hospital
Harvard Medical School
Richard B. Simches Research Center
185 Cambridge St., CPZN 5.612A
Boston, MA 02114
Ph. 617-643-5536
Fax 617-726-5735
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