Transgene Preparation for Microinjection

The quality of your DNA is important for the outcome of your transgenic mice.
Please follow these guidelines in preparation of your transgene for microinjection.

1. It is essential that you remove the transgene from the plasmid backbone leaving as little plasmid sequence as possible. It has been shown that residual plasmid sequence will decrease integration and expression of the transgene. Please make sure the plasmid stock you are using is free from bacterial genomic DNA contamination.
2. Isolate the fragment on a fresh TAE agarose gel using high quality agarose. Borate ions are toxic to eggs (no TBE gels) and heavy metal contaminants of lower grades of agarose are toxic, too. Sea-Chem GTG agarose (Fisher # BMA 50070) is recommended.
3. Cut the gel slice out of the gel quickly, as UV radiation will nick your DNA fragment. The Qiagen kit Qiaex II (Qiagen # 20021) is recommended for removing the DNA from the gel. Resuspend in sterile TE (10mmol Tris/ 1 mmol EDTA pH7.4) at a concentration of 30-100ng/ul. About 20 ul of concentrated stock is need for injection.
4. Bring your DNA and completed requisition form to 5046 Malott. The form should be filled out entirely and include a picture of the gel. Along with your purified fragment, please run a fragment of known concentration, cut or uncut plasmid, and a DNA size marker.
5. Once the transgene and completed form are received, you will be notified of your tentative injection day.
6. I can be contacted at (785) 864-4537 or by email at dbrobst@ku.edu if you have any questions.