[Zbrafish] Re: DNA preps for injections

Wilson Clements via zbrafish%40net.bio.net (by wilson.clements from gmail.com)
Wed Dec 17 18:55:04 EST 2008


Dear Michelle,

For injection of DNA, I use regular old minipreps (Qiagen, Eppendorf,  
whatever) or midipreps.  Then I phenol extract (add equal volume of  
24:25:1 phenol:chloroform:isoamyl alcohol, vortex, spin 3', pull off  
aqueous (top) phase) them twice and ethanol precipitate (add 10% vol.  
of 3M sodium acetate, pH 5.2, mix, add 2.5 vols. 100% ethanol, mix,  
-20C 15' to overnight, spin full speed 15' at 4C, pull off supe, and  
resuspend pellet in appropriate vol. H2O).  I have no problems with  
injected DNA cleaned up in this manner and the clean-up is cheap and  
amenable to reasonably high throughput.

For BACs, we set up 5ml o/n cultures.  We spin down the cultures,  
resuspend them in Qiagen midiprep buffer P1, lyse them with P2 (5',  
r.t.), neutralize with P3, spin down the garbage, transfer the supe  
(~750ul) to a new tube, add equal vol. isopropanol, spin 30' at 4C,  
wash with 70% EtOH, and resuspend in 50ul H2O.  This prep is pretty  
dirty, but can be further cleaned up with phenol extraction and EtOH  
precipitation.

Hope this helps.

Best,
Wilson
------------------------------------------------------------------------ 
--------------------
Wilson Clements, Ph.D.

wclements from ucsd.edu

Dept. of Biology
Section of Cell and Developmental Biology
University of California at San Diego
9500 Gilman Dr.
Natural Sciences Building 6105
La Jolla, CA 92093-0380

TEL    (858) 534-6955
LAB    (858) 822-4658
FAX    (858) 822-5740


Message: 1
Date: Tue, 16 Dec 2008 18:01:51 -0500
From: Michelle Emond <emond.4 from osu.edu>
Subject: [Zbrafish] DNA preps for injections
To: zbrafish from magpie.bio.indiana.edu
Message-ID: <4948335F.2000800 from osu.edu>
Content-Type: text/plain; charset=ISO-8859-1; format=flowed

Hi,

We've been trying to find a reliable kit for cleaning up DNA for
injections.  Qiagen miniprep DNA does not seem to be clean enough to
inject straight off the column, as the embryo survival rate is very
low.  So, we've been running the DNA over one of their PCR purification
columns to further "clean" the DNA, and that seems to result in higher
survival rates.  However, we'd like to streamline our methods to cut  
costs.

We'd love to hear what other labs do for DNA preps (plasmids and BACs),
both for larger preps of known constructs (midi or maxi), and for
miniprep quantities for screening potential constructs.

Thanks very much,
Michelle Emond, Ph.D.
email:  emond.4 from osu.edu
Center for Molecular Neurobiology
Ohio State University




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