C.B.S. Scientific GM-2000 - Manual (Page 8)

C.B.S. œ Scientific Linear Gradient Maker 8
3.2 General
Notes
In general, a gradient maker consists of two cylindrical chambers, with connecting channel
between them. The two chambers may be arranged side by side or in a concentric arrangement.
Each chamber differs according to which flow outlet or “valve” is connected to it. One flow valve
is connected between the two chambers. The second flow valve is to be connected to tubing.
The gradient gel should be separated into two solutions; each one-half the final volume of the
total gel volume. Each one-half volume should be the high and the low extreme of the gradient.
For example, if the gradient is 20 to 50% UF and the total volume of the gel cast is 30mls, then a
15ml solution of 20% UF and 15mls of 50% UF should be prepared.
Make sure that the output tube is secured (tape) to the gel cast sandwich, ready for pouring. If
the gel is to be poured by gravity, rather than by pumping, close valve #1.
First, identify which chamber is connected to the flow outlet that connects to the output tubing:
this chamber holds the high gradient solution.
Next, ensure that the valve for the flow between the high and low chamber is closed. This valve
should not be open until both solutions are poured. If it is opened before both solutions are
poured, then the volume poured will have one half of its contents flow into the other chamber,
equalizing the volume of solution in each chamber.
Pour one of the solutions into its appropriate chamber.
Flip the valve connecting the two chambers open and close very quickly (without pausing). This
serves to get rid of any air bubbles that may be in the valve.
Pour the other solution into its appropriate chamber.
Open the valve connecting the two chambers.
Turn on the pump that controls the output. If the gel is poured through gravity, then open the
valve on the output tubing.
If you are pouring a 2D gel, do not let the gel pour all the way to the top of the gel cast; otherwise,
there will be no room to load your sample.
If you are doing a 1D gel, it may prove useful to leave 0.75mm of space at the top of the gel and
add a neutral Polyacrylamide plug for the comb.
After pouring the gel, use a syringe to put some buffer at the top of the gel. This allows for the
gel to settle and polymerize with a flat surface for loading. Disconnect the output tubing from the
gel.
If there is excess solution, discard into a beaker through the tubing formerly connected to the gel.
When the gradient maker is empty, add water to clean out the remaining solution and left over
polymerized patches.
After polymerization, transfer to gel apparatus for loading.