DNA Purification for transgenic microinjection
(modified 2008 Feb 27)
Take extreme care to minimize particulate matter or other contaminants in the linearized
gene fragment solution. Prepare at least 50 ul of a 100 ng/ul solution.
1.
After cloning your gene construct, purify the vector using, for example,
Qiagen column, or Cat. No. 11451-028, MARLIGEN per plasmid DNA prep.
2.
Restriction digest the vector, separating the fragment for injection.
3.
Run the digested DNA on an agarose gel and stain with ethidium bromide.
4.
Visualize with longwave uv light to avoid degradation and excise the
band containing the gene construct.
5.
To free the DNA from the agarose use the QIA Quick gel extraction kit, Cat.
No. 28704 sold by Qiagen, according to the manufacturer’s protocol.
6.
Add 1/10 volume of 3M Acetate and ethanol precipitate in 2-2.5 volumes of
absolute ethanol. Resuspend in elutip buffer.
7.
Pass the DNA through an elutip-D-mini-column sold by Schleicher
and Schuell, Cat. No. 10462615/ E04345, following the protocol from the
manufacturer.
8.
Ethanol precipitate and wash well with 70% ethanol. Be careful to evaporate
the ethanol completely, but do not use a speed-vac. Resuspend your DNA in filter
sterile TE (10 mM Tris, 0.1 mM EDTA, pH 7.4) from the Transgene Facility at
20-100 ng/ul. The Facility needs approximately 1,000 ng of purified DNA.
9.
Spin in a microfuge for 15 minutes at 14,000 rpm and then pipette off
the supernatant containing the DNA for injections.
10. Besides DNA, bring the Transgenic Facility a picture of a gel documenting the
purity of the fragment and an estimate of the DNA concentration.