Research

Transgenic Services: Protocols

Transgenic Services

Preparation of DNA Fragments for Microinjection

The preparation of the DNA is critical to the success of the transgenic experiments. The DNA must be (1) free of salts, organic solvents, traces of agarose, and micro-particulates (2) in the correct sterile, filtered buffer and (3) not sheared or nicked.

Microinjection Buffer (TE-inj):

10 mM Tric-HCl, pH 7.4
0.1 mM EDTA (this lower concentration is important as EDTA is toxic to embryos)
autoclaved and filter (0.2 um) sterilized

A large prep of the plasmid DNA can be made and purified by cesium chloride CsCl) gradient or a Qiagen column.

Isolate the fragment from vector sequences by restriction enzyme digestion, removing as much of the vector sequences as possible.

Separate the digested DNA fragments in an agarose gel using 1X TAE buffer containing ethidium bromide (0.5 ug/ml.

View the DNA fragments using a long-wave UV light and carefully excise the fragment of interest using a fresh, sterile scalpel. Minimize exposure to UV. Dice the agarose block and transfer to one or more 1.5 ml eppendorf tubes depending on the size of the gel slices.

Clean the DNA fragment by using a Qiagen QiaexII gel extraction kit (Qiagen catalog #20051) or Geneclean Kit (Qbiogene catalog #1001-200). Elute DNA with TE-inj (above).

Quantitate the DNA concentration by either a fluorometer with calf thymus standards or by comparing an aliquot of the DNA prep with size standards of known concentration on an agarose gel.

Give a total of 1 ug DNA at 50 Ng/ul or more in an eppendorf tube to the facility for microinjection. Attach a picture of an agarose gel showing an aliquot of the purified DNA fragment and its concentration (include size and concentration standards.

The Transgenic Facility will re-check the DNA prep on a gel; then dilute it to injection concentration, and freeze aliquots at –20 degrees C. Any DNA that does not inject well in test injections, or that performs poorly in in vitro tests; will be returned to the investigator, and a new prep will be required before more injections are performed.

This procedure incorporates aspects of several purification protocols, but is most directly modelled on the Stanford University Transgenic Research Facility’s “Guidelines for DNA fragment preparation for microinjection.”

This page was last updated on: January 31, 2008.