Patricia L. Clark
last updated: April 4, 2002
Preliminary considerations:
* The Gradient Master is pre-programmed for making sucrose gradients in several
different ranges (10-30%, 15-45%, etc). It is easier to use one of the pre-existing
programs than to optimize a novel program; however, if a novel program is required,
the Gradient Master manual has guidelines for program development.
* Gradients should be made as close as possible to the time of use (centrifugation).
Sucrose solutions:
* Make sure you understand the difference between (w/v) and (w/w)!! Most of
our gradients are made with (w/w) sucrose solutions. 100 ml of a 15% (w/w) sucrose
solution will therefore have 15 g of sucrose mixed with 85 g of buffer (not
water! …use whatever buffer your sample is in).
* For high-percentage sucrose solutions, speedy solubilization can be achieved
by gently warming the stirring solution. The old steel stir plates are ideal
for this procedure.
* Sucrose solutions <60% will support bacterial growth over time. To combat
this, filter sterilize your solutions, and keep them in the cold room. Do not
autoclave.
Loading tubes with sucrose
solutions:
1. Decide whether to use the SW28 (shorter, fatter) or SW28.1 (longer, thinner)
rotor and tubes. Make an even number of gradients, so the rotor will be balanced
(or make 3).
2. Mark tubes using the aluminum gig provided for each tube size: stick tube
in gig, make ultra-fine tip sharpie mark around tube using either upper or lower
edge of gig. Upper edge is for smaller caps/smaller sample volume; lower is
for large caps/samples.
3. Pipet lower percentage sucrose solution into tubes, ~1 mm above mark (does
not need to be exact, or equivalent to other tubes).
4. Use syringe with cannula to layer higher percentage sucrose solution under
lower percentage: fill syringe with solution, slide cannula down inside edge
of tube until hit bottom of tube, gently displace lower percentage solution
with higher. Keep adding sucrose until boundary is exactly at gig mark. Remove
cannula.
5. Put caps (small or large) on tubes: find overflow valve on cap. Push cap
onto tube, with the valve side slightly higher, so the air and excess sucrose
escapes through the valve. There should be no air between the cap and the solution.
Blot excess solution in cap center with kimwipe.
Gradient Master procedure:
1. Turn on Master. Check if platform is level (use bubble level). If not, use
dial on control panel and thumbscrew next to control panel to adjust. When level,
press ‘DONE’.
2. Press ‘GRAD’ to enter the gradient menu.
3. ‘LAST’ will display the last gradient used. ‘RCNT’ will
display recently used gradients, and ‘LIST’ will display the full
list of gradients. If using ‘RCNT’ or ‘LIST’, select the
rotor you will be using (SW 28 or 28.1).
4. Turning the control knob will scroll through the available gradient programs.
Turning the knob more will increase the scrolling speed.
5. Gradients are listed by:
...........Short/Long: this refers to the caps on the gradients
...........Sucr: sucrose
...........% range: many ranges are pre-programmed
...........1/2St: refers to one- or two-step gradient forming program
6. For example, if you want to separate a 200 ul sample on a 15-45% sucrose
gradient using the SW28.1 rotor, you would select “Short Sucr 15-45% 1St"
. When you find the correct program, press ‘USE’.
7. The screen will show a summary of the run program, including the name of
the gradient, the first and total number of steps, and the time and angle used
for the first step.
8. Place appropriate tube holder (magnetic base) on platform; put tubes with
caps in holder.
9. Press ‘RUN’.