PREPARING DNA FOR MICROINJECTION
Digest at least 10ug (but up to 50ug) of maxiprep plasmid with suitable enzymes (for at least
2 hours).
Check digest (run a small amount on to gel to check it is cut properly).
Run on 0.7% agarose gel (conc of gel is kept low so that agarose contamination is
minimised, for the same reason it is good if the gel is overloaded).
Cut out the required band and purify with Qiagen gel purification system.
Elute DNA with 60 ul transgenic injection buffer (5-10mM Tris-HCl pH 7.4, 0.1-0.25mM EDTA
– normal TE is not suitable).
To calculate concentration of DNA for injection (it is important that DNA be very accurately
quantitated):
Using a square of plastic (say 10cmx10cm cut from a clear plastic bag) aliquot out 1µl drops
of standard concentrations of lambda DNA i.e. 25, 20, 15, 10, 7.5, 5, 2.5, 1, 0ng/µl, across
the film. Then place 1µl drop of DNA for injection as well as a 1:10 dilution of DNA. Add 1-2
µl of ethidium bromide (10mg/ml) to 10 mls water and then add 1µl of this mix to each of
the drops on the film.
Sit the plastic on an injection slide for easy transport to transilluminator.
View the drops by placing the plastic onto a transilluminator and measuring the DNA
concentrations from the standards.
Give concentrated DNA to TASQ for injection (do not freeze diluted DNA). Best if diluted just
before microinjection.
It is absolutely crucial that DNA be very pure for injection – ie free of phenol, alcohol,
agarose, enzymes etc – helps keep injected embryos viable. It is also important that DNA be
free of particulate matter that could clog injection pipettes – so usually spin for extended
period (eg 30 mins) just before injection.