The integrity and purity of the transgene DNA used for microinjection are critical for the successful production of transgenic founder mice. DNA impurities (such as bacterial endotoxins and organic contaminants) can decrease the viability of microinjected embryos and the integration efficiency of transgene DNA. Therefore, providing us the highest quality of your DNA construct is very important for microinjection to accomplish your research goals. A number of very useful protocols for DNA purification can be find in Manipulating the Mouse Embryo: A laboratory manual by Andras Nagy et al., 3rd Edition published by Cold Spring Harbor Laboratory Press.
Outline of DNA Fragment Purification:
- Use plasmid DNA that has been purified on a cesium chloride gradient (Sambrook /Fritsch /Maniatis, Molecular Cloning / A laboratory Manual. "Purification of closed circular DNA by equilibrium centrifugation in CsCl-Ethidium Bromide gradients" page 1.42) to obtain DNA of highest purity
- Digest approximately 20~50ug of plasmid DNA with a restriction enzyme that will remove the plasmid sequences from the transgenic DNA construct.
- Fractionate the DNA on an agarose gel and elute the band containing the transgenic DNA to be injected.
- Elute the DNA construct using the gel extraction kit from Zymol Research. Elute DNA using Injection Buffer (Do not use water as it will lyse the eggs). The DNA concentration should be in the range of 0.05 ug to 0.5 ug/ul.
- Determine the DNA concentration and integrity.
|
