[Zbrafish] Re: DNA preps for injections
(by chuntaoyuan from gmail.com)
Tue Dec 23 01:17:04 EST 2008
On 12ÔÂ18ÈÕ, ÉÏÎç7Ê±55·Ö, Wilson Clements <wilson.cleme... from gmail.com> wrote:
> 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
> Hope this helps.
> Wilson Clements, Ph.D.
> wcleme... 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 <emon... from osu.edu>
> Subject: [Zbrafish] DNA preps for injections
> To: zbraf... from magpie.bio.indiana.edu
> Message-ID: <4948335F.2000800 from osu.edu>
> Content-Type: text/plain; charset=ISO-8859-1; format=flowed
> We've been trying to find a reliable kit for cleaning up DNA for
> injections. 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
> 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: emon... from osu.edu
> Center for Molecular Neurobiology
> Ohio State University
Qiagen have different kit for different use. I think Qiagen miniprep
is not so good as Qiagen spin. Qiagen spin is cell grade!
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