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Transgenic Protocol |
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Transgenic / Gene Targeting Facility |
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Purification of DNA for Microinjection 1. Perform a restriction digest to release the gene of interest from vector sequences. 2. Separate the insert of interest from the vector on an agarose gel run in TAE (not TBE). 3. Excise the gel slice containing the gene fragment of interest and elute the DNA. 4. Purify the DNA either by using a DNA purification column or by phenol-chloroform extraction and ethanol precipitation. 5. Final DNA should be dissolved in low EDTA-TE injection buffer (10mM Tris/0.1mM EDTA pH 7.5). This buffer must be prepared with Milli-Q water to maintain zygote viability. We will need 1-2ug of DNA at a concentration of at least 100 ng/ul. 6. Determine the DNA concentration. Leave the DNA in concentrated form in TE injection buffer. 7. The core facility will dilute your sample for injection. |
