Developing Your Own Gradient Run Parameters; Programming The Device - Biocomp GRADIENT MASTER 108 Operating Manual

Press GRAD:
2. If you press LAST, the very last gradient run on the device will appear:
Place your tubes in the holder and press RUN. It is that simple.
3. To recall a gradient that has been used previously, but was not the last one run, press EXIT to return to
the main menu and then press RCNT:
4. When you press the rotor you desire to use, a list of up to 10 gradients will appear in the order of most
recent use. Each time you use a gradient for a given rotor, it goes on top of the list and the other
gradients shift down one position. Use the DOWN and UP keys to scroll through the list as you did in
LIST. When you find the gradient you desire to run, press USE and RUN to begin making the
gradient.
F. DEVELOPING YOUR OWN GRADIENT RUN PARAMETERS
Although you are supplied with a number of gradient profiles, you may find that the gradient you
desire is not on the list. Here we describe the method we use to develop the parameters to enable you
to do the same. First, it is essential that the heavy solution be dyed so that you know when to stop
mixing and so you can evaluate the gradient's shape either visually or with a flow cell. We have had
success with 1-2 ml 0.4% Trypan Blue/100 ml solution or 1-4 ml 0.34% Coomassie Brilliant Blue R/100
ml. (See Coombs and Watts. 1985 Anal. Biochem. 148; 254). For salt gradients (CsCl, NaCl or K
Acetate), use Bromphenol (not bromophenol) blue. India Ink (carbon black) works well in high solute
concentrations since it does not change color. It does tend to aggregate and settle over time, however.
In developing new run parameters, you should be guided by the values in the gradients stored in
memory or at the back of this manual. There is probably already a gradient there close to the one you
plan to make, and you should start by using its listed angle and speed. Time is then the dependent
variable which you will determine empirically, counting up from 0:00 until the gradient is finished.
Should this strategy fail, minor variations in angle and speed will usually produce the gradient you
seek. Remember: the goal is to cease gradient mixing when the first traces of blue dye in the heavy
solution reach the upper corner of the light solution next to the cap.
G. PROGRAMMING THE DEVICE
1. Press NEW:
GRADIENT MENU
LAST RCNT LIST NEW
Short Sucr 02-28% wv 2St
RUN SW50
Select Rotor, Use Arrow
SW28 28.1 SW41 SW40 EXIT
GRADIENT MENU
LAST RCNT LIST NEW
Select Rotor, Use Arrow
<--> 28.1 SW41 SW40 EXIT
13
EXIT
EXIT
EXIT