DNA Prep for microinjection
Plasmids should
be purified using an endoplasmid free kit such as this.
It’s important to
excise your transgene from the plasmid reducing plasmid DNA as much as possible.
It’s also important to submit DNA that is free from impurities that could clog
the injection pipets or kill the injected embryos. The following 2 kits are popular
methods for preparing DNA for pronuclear microinjection:
QIAquick Gel Extraction Kit (70 bp – 10 kb)
QIAEX
II Gel Extraction Kit (40 bp – 50 kb)
With both kits,
use T-lowE (10
mM TRIS, 0.1 mM EDTA) which is available from the Transgenic Core for the final
elution step.
Even with kits, we see
variation in the purity of the DNA submitted. The purity of the DNA and the
accuracy of the quantification are the 2 most important factors in the success
of your injections. Even when using a kit, careful attention to details is
essential! Follow all extra steps for increased purity. The goal is to obtain
4ug DNA at 100ng/ul.
For those who are opposed to
the use of kits, here is an additional protocol: