I have stained cells with DAPI (20 ug/ml) for 5 min at room temperature.
Cells were fixed by adding ice-cold methanol preferably at -10 C for 5
min. Cells were rinsed with TBS twice before staining with DAPI. Wash
cells with PBS several times before imaging. 
You may want to see Betapudi et al. (2005) MBC.16: 2248-2262. 
Hope this will help you
Venk
--------------------------
Venkaiah Betapudi, Ph.D.
Assistant Professor of Molecular Medicine
Department of Cell Biology (NC10)
Cleveland Clinic Foundation
9500 Euclid Avenue
Cleveland, OH 44195
Tel 216-445-2359
Fax 216-444-9404
E-mail [log in to unmask]
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-----Original Message-----
From: DICTY [mailto:[log in to unmask]] On Behalf Of
Kari Naylor
Sent: Thursday, November 10, 2011 10:40 AM
To: [log in to unmask]
Subject: [DICTY - 692] two questions

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.  


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???


Thanks in advance

Kari

Kari Naylor PhD
University of Central Arkansas
Biology Department
139 Lewis Science Center
ph:501-450-5826
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