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
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
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).
Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT
(t) +44 (20) 76797264 (f) +44 (20) 76797805 (e) [Only registered users see links. ]
I haven't injected DNA, but I have injected antibodies into cells year
back. We used to give them a brief (about 20 min) spin at about 100,000
X g in a Beckman Airfuge (sort of a Barbie-sized ultracentrifuge driven
by compressed air).
Nick Theodorakis [Only registered users see links. ]
contact form: [Only registered users see links. ]
In article <firstname.lastname@example.org. com>, "Nick Theodorakis" <[Only registered users see links. ]> wrote:
Standard practice in many techniques. Spin down everything insoluble
and aggregated. Do they still make Airfuges? The greatest thing about
airfuge is that its rotor levitates. Great idea that works exceedingly well!