A Work in Progress (last updated 11/22/2007):
SORRY, THE PCR MACHINE IS NOT HOOKED UP YET.
The virtual laboratory is still under development, as is the
user documentation. These are brief notes on how to use the
virtual lab for restriction digestion and electrophoresis,
and some suggested experiments. For background and a more
detailed protocol, see the Phase I lab manual,
http://www.attotron.com/cybertory/CybertoryManualAA3.pdf
Questions or Comments:
Please contact Dr. Horton (rmhorton@attotron.com) if you are
interested in beta testing this software in classes, or if
you have any questions, comments, or suggestions.
Using the Interface:
Click the tip box to put a tip on the pipette if it doesn't
already have one.
To throw a tip away, click on the opening to the waste can.
Alternatively, click the tip ejector on the pipette.
To move the pipette to a tube, click the tube. The top
should pop open, and the pipette will fly over if it has a
tip. The pipette will not go to the selected tube if it does
not have a tip. You have to click a tube WITHOUT DRAGGING to
select/deselect it. If you drag a tube, it will not be
selected. The selected tube (with its top open) can not be
dragged; if you want to move it, deselect it first.
Click the pipette plunger to toggle it up and down. Select
the pipette volume by dragging across it with the mouse, and
type in a new volume. Only volumes between 0 and 100 ul are
valid. Alternatively, you can click on the left and right
sides of the volume dial wheel to change the volume.
To see how much solution is in the pipette tip, you can move
the pipette to the pipette stand, either by deselecting the
tube, or by clicking on the pipette stand. (You can also
drag the pipette out of the tube, but I will probably
disable this, because it is confusing to be able to drag the
pipette when that is NOT how you get it into a tube. It is
also a pain to get it to go in front of or behind all the
right things.) (There is also a bug that sends the pipette
off into space after it has gone to the pipette stand
because its tube was deselected.)
To see how much solution is in a tube, zoom in. Drag the
tube a tad so it pops in front of the tube rack (drag by the
cap hinge, so the hand doesn't block the label). It will
snap back in place when you let go.
To measure how much solution is in a tube, try to remove
more than you have with the pipette. The pipette volume will
only take up what is available, and the pipette volume be
set to the amount drawn up.
Only one tube can be selected at a time. Choosing a
different tube will open its top, and close the top of the
previously selected tube.
Drag tubes around to move them. You can only place tubes in
the holes of tube racks and the PCR machine. If you try to
drop a tube anywhere else, it snaps back to its former
position.
To throw a tube away, drag it to the waste can. To get a new
tube, drag it from the tube jar. (NOTE: tubes from the tube
jar act differently then the original tubes, because they
have not been assigned a position yet. You can drop them in
mid-air, for example. They are also frozen (-120 degrees!).
I will probably change this behavior, so they go back to the
tube jar unless you drop them on a valid position. Once you
drop a tube from the tube jar onto a valid tube position, it
behaves normally (i.e., won't hang in mid-air any more.)
tubes take their temperatures from their positions. The
water bath is 37C, the ice bucket is 0C, and room
temperature is 20C (25C is more comfortable, but I wanted to
exaggerate the difference from the waterbath.)
Solutions in tubes are ALWAYS incubating. Nothing happens if
the tube is at zero degrees (or below). Enzyme activity is
temperature dependent; thye cut best at 37C, but will also
cut slowly at room temperature. Enzymes also degrade in a
temperature dependent fashion. The lengths of the
incubations are measured by the digital clock on the wall.
To speed up incubations, hit fast forward on the clock. You
can also cheat and stop the clock to set up reactions if you
like. (For now, the clock only controls incubation times;
the PCR machine and gel box keep their own time. I plan to
hook the whole universe up to the clock eventually. Enzyme
activity should also depend on pH, ionic strength, [Mg++],
etc., but it doesn't yet. These enzymes will cut just fine
in plain water, thank you.)
To load the gel, suck up a solution into the pipette, then
click on the target well. The pipette will fly over.
Use Ctl-click (Mac) or right click (Windows) to bring up the
Flash menu. This will let you zoom in. It is much easier to
read the tube labels and see the gel wells if you zoom in.
The tubes are labeled (with up to three letters).
buf: Universal 10X restriction enzyme buffer
Bsu: Bsu36I - the enzyme whose site is affected by
the sickle cell mutation.
Eco: Eco RI
Hin: HindIII
H2O: water
SS: homozygous sickle cell control
WW: homozygous "wild type" (normal) control
Pt1, Pt2, Pt3, Pt4: patients to be diagnosed.
Sample Protocol:
* Mix the following reactions:
- Tube "1a": 100ul water, 20 ul 10X buffer, 80 ul "SS"
DNA (for a total of 200ul).
- Move 100ul to tube "1b".
- Add 5ul Bsu36I to tube "1b".
* Place both reaction tubes in the waterbath for 30 minutes
(fast forward the clock).
* Load the incubated solutions into gel wells 1 and 2.
* Click the power button on the electrophoresis power supply
to run the gel.
You only get to load and run the gel once (though you can
turn the power on and off as many times as you like once the
gel is loaded). To start over, press the reload button on
your browser. You will have to log in again.
Suggestions of Things to Experiment With:
* Can you see a difference between "WW" and "SS" when cut
with Bsu36I?
* Which patients are normal? Which have the disease? Which
are carriers?
* What about the other enzymes?
* What happens when you put in multiple enzymes?
* How much enzyme does it take to achieve complete
digestion? How much time? How much time at room temperature?
* How long does it take to ruin an enzyme by letting it sit
around at room temperature? How long at 37 degrees? (ANSWER:
you can't ruin enzymes yet, but you will be able to soon).
* How can you recognize partial digestion?
* (BONUS!) Construct a restriction map of the SS and WW
DNAs, using the enzymes provided. (We'll need a molecular
weight marker for this...)