Tutorial
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amount of solution loaded into the micropipette. Compare the two methods
outlined below.
1) 1
µ
g of plasmid DNA is added to 5 ml of a standard physiological salt
solution; a DNA concentration the same as for most standard lipofection
techniques. 2
µ
l is used to load the micropipette.
2) 1
µ
l of plasmid DNA (1
µ
g/
µ
l) is added to 29
µ
l of standard physiological
salt solution.
Both of these techniques use DNA sparingly. In the former, the total DNA in
the pipette is about 0.4 ng whereas in the latter it is 67 ng.
Of course, the filling solution need not contain genes. This technique should
work for electroporating small peptides, drugs, dyes, calcium ions and probably
even a variety of uncharged molecules. Filling is generally done in two stages.
1. Use a hand-held pipetter with a 10 µl disposable tip. Add 2 to 3 µl of
the solution to be electroporated to the back of the micropipette. The
solution moves along the internal glass filament by capillary forces and
fills the tip. It may take a few taps of the glass to get the flow started.
After 30 seconds or so, apply a few gentle taps near the shank to
dislodge bubbles.
2. Use a 1 ml syringe to backfill the micropipette with the solution used
to dissolve the molecules to be electroporated. You should start adding
the solution several millimeters behind the tip and fill to about the
middle of the pipette length. Gently tapping the micropipette shank
will dislodge the air bubble behind the tip.
Needle-tubing combinations that work with standard syringes are available
from commercial suppliers such as:
http://www.smallparts.com/components/
http://www.wpiinc.com/WPI_Web/Lab/MicroFil.html
Tutorial
