Cleaning up DNA for microinjection

[Tom Anderson]

Hi everyone,

Back with another purification question. I'm trying to do some DNA
microinjection into cells, but am finding that i'm not getting flow
through my needles. When i use some control DNA from a colleague, it works
fine, so it seems my DNA solution is too lumpy, and is clogging the
needle.

So, any ideas how i can remove particulate matter from my DNA?

The DNA is originally from a Qiagen prep, and so far i've tried spinning
it down and putting it through a 0.22 micron cellulose acetate spin
filter, but neither of these solve the problem.

How about putting it through a silica column, such as a QIAquick? In
theory, the particles should either get stuck in the membrane or get
eluted in the washes, before the DNA. However, i am aware that silica
fines can leach from the membrane, which might also clog my tip.

Would CsCl purification work? Presumably, particulates won't band with the
DNA.

I could perhaps filter through an even small spin filter - since my
construct is ~3 kbp, it might well go through a 500 kDa filter. This could
be quite expensive, though.

Perhaps a fresh DNA prep is the way to go!

Also, does anyone know what the typical diameter of a microinjection
needle is? I know they vary according to how you prepare them, but i don't
even know what order of magnitude i'm working at. All the information i
can find pertains to the needles used for injecting oocytes, which seem to
be much larger (several microns across).

Many thanks,
tom

-- 
Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT
(t) +44 (20) 76797264   (f) +44 (20) 76797805   (e) thomas.anderson At ucl.ac.uk