Preparation of DNA

For the chromosomal integration and germline transmission of transgenes, the DNA should be linearized or isolated from the plasmid sequences. Supercoiled DNA, however, can be used for transient gene expression work. After phenol/chloroform extraction to remove the restriction enzyme, the DNA is further purified on a microconcentrator (Microcon 100, Amicon Inc., Bedford, MA) as follows:

1. Equilibrate the microcon with 250 ||L sterile dH2O and spin at 3000 rpm for 5 min.

2. Load the DNA solution into the microcon, and spin at 3000 rpm for 5-10 min until most of the liquid has gone through the membrane.

3. Wash the microcon three times each with 500 |L sterile dH2O, and spin as above.

4. Invert the microcon, and spin briefly to recover the DNA solution into a fresh Eppendorf tube. Estimate the DNA concentration by running an aliquot of the purified DNA on an agarose gel, and store the DNA at -20°C.

5. Before microinjection, dilute the DNA to 0.1 |L in 0.2 M KCl, 0.25% phenol red. The dye facilitates the estimation of the injection volume.

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