- Start the DNA purification with a high quality plasmid DNA preparation which does not have any bacterial DNA contamination.
- Digest the DNA so that the target insert is cut out. DNA used for DNA microinjection must not contain any plasmid DNA (foot note1).
- Check with a small aliquot that the digestion is complete and then run a preparative gel where the insert and plasmid fragments are well separated. We wish to receive a picture of the gel with your DNA microinjection order. Try to use as little UV illumination as possible since UV light causes single strand breaks (footnote 2) .
- Cut out the insert and purify the DNA with a DNA purification kit. Qiagen II -extraction kit works fine but other kits might be as good. The DNA can be dissolved at this point in the injection buffer or it can still be continued to be purified with 2-propanol precipitation and further by dialysis against the injection buffer.
- In both cases the DNA must be finally dissolved in the injection buffer: 10 mM Tris, 0,1 mM EDTA, pH 7,4.
- Concentration and quality should be checked in an agarose gel and we would again like to receive the picture.
- We would like to receive about 100-200 microliters of the DNA with a concentration of 20 ng/ microliter. For injections we further dilute the DNA to 2-5 ng/microliter.
Problems in purification might result in no transgenic mice or a small number of transgenic mice.
Footnote 1: If plasmid DNA is included in the injected DNA, it will be methylated in the cell and the transgene will be silenced.
Footnote 2: Small single strand breaks caused by UV illumination cannot be detected by agarose gel electroforesis. Fragmented DNA is not incorporated into genomic DNA and the result is a small number of transgenic mice.
Last updated: 2.2.2012