Preparing And Capping The Gradient Tubes - Biocomp GRADIENT MASTER 108 Operating Manual

through the list.
When you have found the gradient you desire, press the USE key
Press the DOWN or UP keys to scroll through the steps in this gradient. Press the RUN key when you
are ready to start the run.
A note on steps: Most gradients require mixing at different times/angles/speeds to produce the
desired shape of the gradient. Each step has its own time/angle/speed and they are run in sequence
automatically when you press the RUN key.
C. PREPARING AND CAPPING THE GRADIENT TUBES
1. Introduction. One of the problems we encountered was the wide variation in the capacity of tubes
from different manufacturers, and even in lots from the same source. To avoid problems with specific
volumes in gradient recipes, we now supply each tube holder with a Marker Block which marks the
tube's half full point regardless of source or lot variations. Since you have the choice of using the short
cap for small sample, velocity (rate zonal) runs or the long cap for the large samples used in isopycnic
runs, we now provide a stepped Marker Block to suit both the 10 mm and nm cap types in the open-
top swinging bucket tubes.
Note that the gradient run parameters provided with the short caps apply ONLY to gradients run
with the short cap and vice versa.
Half-full mark for short, 4mm cap
Half-full mark for long, 10mm cap
2 Step Marker Block
2. Before filling the tubes, place them bottom down into the appropriate Marker Block and use the fine
tip marker provided to make a small "Half-full" mark on the tube where it meets the top of the
block. Use the upper step for the nm cap and the lower step for the 10 mm cap as illustrated above.
This will be your filling guide during layering of the heavy and light solutions.
3. Attach the two cannula provided to syringes and fill all tubes with filtered, degassed, room
temperature light solution ~nm above the half-full mark. Filtering with Whatman #1 just before use is
usually enough to degas as well. With practice, you will see just how much extra light solution to add
so that only a small amount is displaced into the cap. Accuracy is not crucial , in fact you may find it
easier to simply pour the light solution in, especially with the larger volume tubes. However, you
should be careful to avoid leaving bubbles adhering to the wall of the tube.
4. Fill a syringe with filtered, degassed, room temperature heavy solution and insert the cannula quickly
to the bottom of the tube and begin layering the heavy solution. Holding the tube at eye level,
carefully fill until the heavy-light interface rises precisely to the half-full mark. If the tip of the cannula
is kept just under the light interface as it rises, you can layer faster without disturbing the light
solution. Withdraw the syringe quickly and smoothly and then add or remove light solution so that
there is enough to fill air hole when the cap is inserted.
00-50% S1/2 5:00/60.0/25
RUN DOWN
Rate zonal runs
Isopycnic runs
UP EXIT
Insert tube and make mark with
fine tip marker on the tube at the
appropriate step in the marker block
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