Inventory of stocks in the lab:
Aliquoting UPenn stocks
Gordon Smith's way:
I use 4 uL aliquots. I like 1.5 mL tubes, they sit in boxes nicely, and 4 uL will sit at the bottom of the tube nicely. I use 10 µL extended length barrier tips and P10 for all the virus work (a P20 would also do the trick, just make sure you use barrier tips).
I thaw the UPenn stock on ice, vortex very briefly, and pulse spin.
I then will aliquot, usually only 4 tubes at a time, returning the stock to ice in between (this way there isn't too much time any one aliquot is sitting at RT). After making each set of 4 aliquots, I snap freeze them in a dry ice, 100% EtOH slurry (you could use liquid nitrogen, but I've been told it may be a little too cold / too fast). I also flick the stock with my finger a few times before starting each set of 4 aliquots, just to make sure the stock is well mixed.
Some of this is probably overkill for AAV, but I started out using HSV, which is not very stable in the absence of a host. It's been working great, so I see no need to change. I also usually aliquot in a sterile TC hood if there's one available.
Sharon Huang's way:
Yes, we aliquot the virus. We use centrifuge tubes or smaller tubes, and I usually put 5 ul in one tube, because I inject abut 2 ul each time. Gordon put 3 ul to each tube, and he use 0.5 ul or 1 ul each time. We then store the tubes with virus to -80.
When to get the virus: Again, this is from my HSV days (which I think is a lot less stable), but I actually keep it in the -80 until I am ready to inject. I prepare the craniotomy and duratomy, then load and inject. While this does require re-scrubbing in afte the injection, I think it gives me the best chance of success.
Actual injection: I usually go with 18 pulses at 32.2 nL each (total vol 580nL), with 15 sec between pulses. I usually hold the pipette in place for 5 min before withdrawing. Because I'm always uncertain of exactly when I contact the brain (due to the CSF) I usually repeat this at 2 depths, the first around 500 µm down, and the second around 250. I like doing the deeper one first.
we're using a little fast green (just enough to color the solution) in the injection, it helps make it clear that the tip is in the brain and helps gauge how much leakage / backflow there is around the tip.
Re: fast green. I actually haven't added it to the aliquots. I was nervous at first about messing up the entire batch. It seems to be working fine, so I guess you could do that. What I actually did was make a concentrated solution in saline (i didn't measure, but it's very dark but completely dissolved -- i really should filter it to make sure there are no clumps, but I haven't yet).
At the time of the injection, I flick this green tube so a few tiny drops get scattered on the sides. I then touch the tip of the pipette to a smallish drop, then put it into the tube with the freshly thawed viral aliquot. I mix just by stirring with the pipette tip (you never want to pipette the virus up and down). This way the virus concentration is barely changed. I tried it with dissolving the powder directly in the aliquot, but I couldn't get it to dissolve completely, and it kept clogging injection pipettes.
Not very precise, but it's been ok. For some injections I want it really dark (if i'm doing tiny injections), but for the GCamP-level volumes lighter is better because it gives a better chance of seeing each pulse come into the brain.
It's F7258 from sigma, the fast green fcf.
[We make a nick in the dura.] Also, if it comes to a choice of larger craniotomy and duratomy versus not being sure I have a clear path to cortex and a good penetration of the pipette, go with a larger opening. It usually heals up well for me.