Hi all, this may be a stupid question but I was wondering how/if people store their DNA mixtures for microinjection. There’s a mixture of opinion in my lab. Some make it fresh each time, others store it at -20ºC for a few days, etc. I’m doing a lot of injections of a particular construct and am trying to balance convenience (ie. making one mix and using repeatedly) vs. injection efficiency. All DNA is midiprepped, miniprepped with a good kit (ie. Invitrogen), or EtOH precipitated. The injection solutions are all ran through a filter to remove particulates. Thoughts/opinions are greatly appreciated.
I store my injection mixes at 4°C for days, weeks, months, or even years. No issues, just respin before you load a fresh needle. I was advised to never freeze the mixes, and our mix has PEG and some other goodies. DNA in other solutions might be better stored in other ways.
OK, great to know. I had been storing my mixes at 4ºC and then had a particular injection start failing. I had wondered about the storage temp causing issues. Thanks!
Long-term storage is not typically an issue because you shouldn’t typically need to recreate a transgene (it’s far easier to freeze a good line than to make one, and often better to mate a transgene into a new background instead of injecting), plus you often move on to new constructs and new targets.
Still, I’ve a tube of injection mix that I’ve used to teach a few different people to inject, years apart, stored at -20 in between uses. I don’t see why a few freeze-thaws would be a big problem for a DNA microinjection mix. I’ve never filtered to remove precipitates, just spun at maximum speed in the microfuge for 5+ minutes before loading the needles.
There’s also the question of whether you need microinjection buffer; I was taught to use it in microinjections, but have since discovered other people just inject DNA without it, and get their transgenes just fine.
Re: on mating extrachromosomal transgenes vs. making a new one.
While I would defer to Hillel on just about anything, I hate mating extrachromosomal transgenes. My understanding is that the arrays are treated like an extra-X chromosome during meiosis. As a result, it’s easy to mate and create males that carry the transgene, getting that transgene out of those males and successfully into a hermaphrodite (requiring the segregation of both an X and an extrachromosomal array) is much less efficient. If your marker is good enough, it should be fine, but the recovery of transgenic hermaphrodites from that cross seems to always be way, way lower than you would like, making recovery of those animals difficult. That said, what’s it’s in there, you’re good.