Kari Naylor wrote:
> Dear Dicty colleagues
>
> I am writing to see if anyone can help us on two disparate issues.
>
> 1) DAPI staining.  We'd love some success stories on DAPI staining.  We've
> fixed the cells as recommended (in 3% glutaldehyde- maybe this is the
> problem) but the staining is quite poor. Any suggestions would be
> appreciated.

Use ProLong Gold Antifade mounting medium containing DAPI from Invitrogen
(P36935). Just make sure to protect from light while the medium cures
overnight and store in a slide box at 4C.

Alternatively, use Prolong Antifade without DAPI and during the 3 third
wash add Hoescht 33342 at 1:5000 dilution in PBS ( I wash cells 6X after
the secondary). Again, make sure to protect from light even during the
remaining washes. I prefer the Hoescht which I also purchase from
Invitrogen (cat. H3570).

>
> 2) we are trying to make a knockout strain and are having some significant
> issues attaching our up and down stream genomic pieces to BSR gene.  We
> have decided to try pricing out some companies that synthesis large pieces
> of DNA, but to do that we need the entire sequence of the BSR gene (we're
> using pUCBsrdeltaBam).  Does anyone have the complete sequence???

Not sure here, as I do not know what cloning strategy you have tried. So
you haven't had success cloning both targeting arms into the vector or
either of them separately? Sequence could be toxic. Try different cells.
If the plasmid is large and the targeting sequence is AT-rich, try XL10
Gold cells (Stratagene cat. 200314) and grow your transformants at 30C or
lower (even room temp) to reduce re-arrangements of the DNA and toxicity.
SURE cells are decent too, but not so much for large DNAs. XL10 Gold cells
take up large DNAs very well and do not seem to mind Dicty DNAs. Also, why
not try another vector like the pLPBLP (Faix et al, 2004) or if you are
concerned about the Cre sites or cut out the Bsr cassette and insert it
into another vector.

Genescript and Geneart will synthesize DNAs of various sizes...I have had
500bp to 12,000bp synthesized using GeneArt...quick turn around,
affordable, optimized sequence for E. coli and also provides the option of
optimizing long trinucleotide repeats that can be problematic or altering
codon bias.

Mike

>
> Thanks in advance
>
> Kari
>
> Kari Naylor PhD
> University of Central Arkansas
> Biology Department
> 139 Lewis Science Center
> ph:501-450-5826
> [log in to unmask]
>


----------------------------------------
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
[log in to unmask]
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