Instructions for How to Setup the Robot to Print
Introduction
This document provides guidelines for setting up a Pat O. Brown arrayer* to print antigens† onto glass
slides.
Pat Brown’s website, http://cmgm.stanford.edu/pbrown/mguide/index.html, has additional
information about how the arrayer works in general and instructions for how one would build an arrayer
from scratch.
Equipment
Every item has a number so I can reference the equipment throughout this document.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
Print pins (SMP7B)
Sacrificial pins – old pins that are damaged :(
Level
5/32” Allen-wrench
Forceps – Dittmar 300 040
½” wide tape – fisherbrand
Aluminum foil tape – Scotch Brand 425-2322-10-11
SME Slides “good”‡ – 5 boxes of 25 per box
Poly-L Lysine Slides “sacrificial”§ – 12 + 1 or 2 for the test print
ddI** H2O ~250 mL
1X PBS
20 µL pipette w/ tips
384-well plate
Dust-Off – air spray for cleaning surfaces
Razor blade or scissors
Foam spacers
Plate Foil – to re-seal used plates
Kapak SealPAK pouches
Layout of the 384 Well Plate
I refer to specific locations within the 384-well plate throughout this document so I wanted to define these
positions to avoid confusion. In the cartoon below, I show 16 of the 384 wells.
The first quadrant contains wells A1 – D4.
The last quadrant contains wells M20 – P24.
*
The arrayer is the name of the complete setup for printing including the robot and platter.
Antigens are a short hand for proteins and peptides.
Good slides are epoxy coated glass slides. As of December, 2003 we are using super epoxy slides (SME) from Telechem Intl.
§
Sacrificial slides are those that should go in the first 6 positions after drawing fluid from the plate (positions 1-6 and 70-75). These
slides receive more fluid so the spots are not as uniform.
**
ddI H2O means double distilled water. This water is of higher purity because it’s been distilled 2x.
†
‡
Checklist for Initial Hardware Setup
Level print head (3, 4)
Clean print table with “Dust-Off” (14)
Add aluminum tape over vacuum block (7, 15)
Fill sonicator with ddI H2O (10)
Secure the vacuum pump to the table
Position the sonicator within its holding pins
Plug in the vacuum pump and sonicator
Turn on arrayer
Put sacrificial pins in print head (2, 5)
Initial Software Setup
Logon to the computer.
Username: scanner
Password: rocdox (as of June 23, 2004, this password was valid. the Relman group keeps the password)
Open the arraymaker250 software.
The startup screen should appear (see below).
Select the Connect tab.
Select Connect to Controller button.
Select the Home all axes button.
This step will take about 45 seconds and the platter should move while the computer communicates with
the arrayer.
The screen shown below should appear when the computer initializing the arrayer.
Select the Align tab.
Align Tab Screen
Motion
Buttons
Control
Buttons
Align
Buttons
Offset
Buttons
Within the Alignment Tab Menu
Move the arrayer by clicking on the motion buttons (forward/back, left/right, & up/down). These directions
correspond to X, Y, and Z respectively. The numbers in the upper left corner under Motion Control
correspond to the current position of the print head (Y & Z) and the platter (X).
Control the distance the arrayer travels by selecting a particular step size. One step corresponds to 1.3 µm
(forward/back & left/right) and 0.6 µm (up/down). For example, when one selects a step size of 20k and
pushes the left button, the print head should move 26,000 µm (~1 inch if one prefers that system of units).
The print head and the platter move independently when moving the arrayer. The print head moves in the
up/down and left/right direction as defined by looking from the front (see Front View of Print Head picture
below). The platter moves in the forward / backward direction only. This motion of the platter is
something to keep in mind when positioning the forward/back coordinates (see Top View of Print Platter
picture below).
Front View of Print Head
The cartoon on the right is a view of the
print head. This cartoon is the view one
would have if he/she were looking
straight on from the front. The arrows
indicate the direction the print head will
move.
Up
Left
Right
Down
Top View of Print Platter
The cartoon shown below is a view of
the print platter.
This cartoon
represents the view one would have if
he/she were looking straight down onto
the platter. The white box is where the
384-well plate goes and the arrows
indicate the direction of movement.
Bias direction
Back
Forward
Align Pins to Various Stations
Important Notes:
•
Use the sacrificial pins for the following steps unless otherwise noted.
•
Make sure the path to the desired location is clear when clicking on a button because the
machine will move after the button is clicked and one wants to avoid collisions (one might
move the arrayer to the X, Y, Z position of the particular station prior to clicking the align
button to verify the path is clear).
•
For the descriptions that appear below, I assume that the arrayer is using 16 pins.
•
I discuss how to deal with different pin numbers at the end of this document.
Motion
Buttons
Control
Buttons
Align
Buttons
Offset
Buttons
The numbers to the right of the align buttons represent the X, Y, & Z coordinate of that station. For
example, in the picture of the align tab screen shown above, the rinse station is located at X = 500000, Y =
-41019, & Z = 86880. Thus, when the Rinse button is clicked, the print head and platter will move to this
location. Additionally, when any align button is clicked, the set button appears. The set button saves the
current X, Y, & Z position. It is important to click the set button after moving the pins to the desired
position.
As an additional word of advice, the X motion control moves the platter only. When aligning the pins to
the Rinse Station and the Dry Station, one can only control the alignment of the Y & Z position with the
software. Careful positioning of the sonicator and vacuum block controls the X alignment (described in the
section on hardware setup).
It is impossible for every slide to sit at the same height. To get around this fact, we can customize the
height at which the pins contact each slide with the offset buttons. Note that we only have to set the pin
offset once. Most likely, and thankfully, someone has already taken care of this procedure for us.
Finally, the control buttons are used to turn on/off the corresponding station so that one can make sure
these devices are functioning properly.
Alignment Checklist
Click the Rinse button (position for sonicator) and align
the tips of the pins to be just below the water level (see
right) in the sonicator (it’s easier to add water to adjust Z
position once the pins are touching the surface)
Click set to save the Rinse alignment
Click the Dry button (position for vacuum block)
Center pins (X & Y) to vacuum block holes
Poke holes in aluminum tape with sacrificial pins (2)
Position the pins (Z) so the edge of the chamfer is just below the aluminum foil
Click set to save the Dry alignment
Replace the sacrificial pins with the good pins (1)
Place the 384-well plate (13) into the print platter using the foam spacers (16) to bias the plate in the
bias direction
Click the Plate button
Center pins (X & Y) on the first quadrant of the print plate (A1 – D4)
Align the Z position (go to bottom of well and back off 700 – 800 steps (~450 µm))
Click set to save the Plate alignment
Click Move to last load (the arrayer should move to the last quadrant)
Center pins (X & Y) on the last quadrant of the print plate (M20 – P24)
Click Calculate correction
Click Save correction
Place a sacrificial slide (9) in the first slide position
Bias the slide in the direction shown to the right and
secure the slide onto the platter with tape (6)
Click the Print button
Align pins (X & Y) with a bias shown in the picture
on the right (for 12 spots in a row with 340 µm
spacing, position X about 3100 steps (~4000 µm) from the bold edge of the glass slide)
Align the pins in the Z position (go down until the pins touch the surface and add another 1000 steps
(~600 µm))
Click set to save the Print alignment
Click Save Alignment to Disk
Select the Test Print tab.
Test Print Tab Screen
Within the Test Print Tab Menu
The test print checks whether the pins are sliding up/down without sticking and delivering liquid at every
spot. If the test print doesn’t look good, then try changing the aberrant pins or doing a wash/dry cycle.
Test Print Checklist
Fill every well in the first quadrant with 7 µL of 1X PBS (11, 12, & 13)
Place the 384-well (13) plate into the print platter making sure to bias it in the bias direction (12, 15)
Place a sacrificial slide (9) in the first slide position
Bias the slide in the direction shown to the right and secure the slide onto the platter with tape (6)
Set the Slide and Plate Setup to the following values (Load Number = 1, Slide Number = 1, Number of
Slides = 1)
Set the Print Setup to the following values (Total number of taps = 144, Number of loads = 1, Sector
Width = 12, Spacing = 340)
Set the Wash and Load Settings to the following values (Wash Cycles = 4, Dry time = 3000, Wash
Time = 4000, Load Time = 1000)
Check and uncheck Bounce Dry, Swish Wash, & Slow Pick Up (it seems necessary to check and
uncheck them a few times for the program to save the correct configuration. In the end, they should be
in the unchecked state).
Click the Start button
Select the Print Run tab.
Print Run Tab Screen
Within the Print Run Tab Menu
This is the final step before turning the machine loose to print thousands of antigens onto glass. Heck the
settings one last time and make sure all the slides are secured on the platter.
Print Run Checklist
Clean the print table with “Dust-Off” (14)
Add the "good" slides (8) to positions 7 – 70 and 77
– 137 (the slides should sit face up as shown to the
right and biased in the bias direction)
Add the "sacrificial" slides (9) to positions 1 – 6 and
71 – 76 (the slides should sit face up as shown to the
right and biased in the bias direction)
Secure all slides to the platter with tape (6) as shown
to the right
Thaw the first print plate (384-well plate full of
antigens)
Vortex the first print plate
Centrifuge the first print plate at 2000 rpm for about
5seconds
Set the Print Setup to the following values (Number
of plates = ?††, Tips = 16, Spots per load = 1, Entire
Platter = check, Re-fill = check, Slow Pick Up =
Uncheck)
††
A typical print uses duplicates of three unique plates (total of six).
Bias direction
Set the Station Settings to the following values (Wash Cycles = 4, Wash time = 4000, Dry time = 3000,
Load Time = 1000 Bounce Dry = uncheck, Swish Wash = check)
Leave the Start Settings as is and make sure Tandem Print = uncheck.
Set the Slide Setup to the following values (Begin on Slide 1, End on Slide 137, Sector Width = 12,
Spacing = 340)
Leave the Blot Pad Settings as is and make sure the Use Blot Pad = Uncheck
Check and uncheck Bounce Dry, Swish Wash, & Slow Pick Up (it seems necessary to check and
uncheck them a few times for the program to save the correct configuration. In the end, they should be
in the unchecked state).
Click the Start button
During the Print
Thaw, vortex, and centrifuge each new print plate.
Sonicate the pins for 3 minutes and then dry for 5 seconds between each print plate.
Remove the plate foil (17) just before putting plate into the platter.
Note: If the pins stick permanently, one can always push stop. The arrayer will stop the next time it goes to
refill. Sometimes a wash/dry cycle will fix things. Other times simply sliding the pin up and down will
work as well.
After the Print
Re-seal the used plate with plate foil (17).
Write on the foil that the plate was printed along with the date it was printed.
Sonicate the pins for 10 minutes and then dry for 10 seconds to thoroughly clean the tips.
Remove all the tape and slides from the platter.
Put the slides back into a slide box and then seal the box + slides in a bag (18).
Store the sealed box in the refrigerator (4oC) until ready to probe.
Put the pins away and clean everything up.
Turn off the arrayer and close arraymaker250.