pcr quality of mouse genomic DNA
ladasky at leland.Stanford.EDU
Tue Nov 12 21:36:16 EST 1996
In article <3282F10E.50A7 at glaxo.com>, Gary Kucera <kucera~gt at glaxo.com> wrote:
>Christophe Andreoli wrote:
>> Dear Friends,
>> I have to prepare mouse genomic DNA, that I am willing to use to clone a
>> about 12 kb fragment by PCR.
>> I am not very experienced in this field, but do you think that a classical
>> extraction would give me a enough clean DNA? What do you think of the
>> Genomic DNA from Quiagen?
>> Thank you
>> christophe Andreoli
>The only problem with phenol/chloroform extraction is the
>mixing involved. If you vortex the sample you will shear
>the DNA into small fragments. This is fine, and often
>advantagous, when PCRing small fragments. But since you are
>interested in a 12 KB fragment you are lowering your chances
>of having a full length template. Instead, I would
>recommend gentle mixing of the phenol/chloroform. A final
>100% chloroform extraction is highly recommended to remove
>traces of phenol.
I prepare mammalian genomic DNA from cell lines routinely. For
all of the mixing steps, including the phenol and chloroform extractions,
I usually flick the side of the Eppendorf tube several times with my
finger. I even tried vortexing once for under a second on a moderate
setting. At the end of the procedure, I always check for DNA fragmenta-
tion by running out an aliquot of the DNA on an agarose gel. I have
always obtained DNA that is larger than 23 Kb. You should be able to
do fairly long PCR from this template... I've done 4.5 Kb using the
Stratagene Taq Extender kit (no affiliations with Stratagene, etc.).
Unique ID : Ladasky, John Joseph Jr.
Title : BA Biochemistry, U.C. Berkeley, 1989 (Ph.D. perhaps 1998???)
Location : Stanford University, Dept. of Structural Biology, Fairchild D-105
Keywords : immunology, music, running, Green
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