Instructions for How to Setup the Robot to Print

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Pierce Bond
6 years ago
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1 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, 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. Print pins (SMP7B) 2. Sacrificial pins old pins that are damaged :( 3. Level 4. 5/32 Allen-wrench 5. Forceps Dittmar ½ wide tape fisherbrand 7. Aluminum foil tape Scotch Brand SME Slides good 5 boxes of 25 per box 9. Poly-L Lysine Slides sacrificial or 2 for the test print 10. ddi ** H 2 O ~250 ml 11. 1X PBS µl pipette w/ tips well plate 14. Dust-Off air spray for cleaning surfaces 15. Razor blade or scissors 16. Foam spacers 17. Plate Foil to re-seal used plates 18. 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 H 2 O means double distilled water. This water is of higher purity because it s been distilled 2x.

2 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 H 2 O (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)

3 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).

4 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.

5 The screen shown below should appear when the computer initializing the arrayer. Select the Align tab. Align Tab Screen Motion Control Align Offset

6 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. Left Up Down Right 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

7 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 Control Align Offset 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 = , Y = , & Z = 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.

8 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 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.

9 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.

10 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 (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 (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) Bias direction A typical print uses duplicates of three unique plates (total of six).

11 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 (4 o C) until ready to probe. Put the pins away and clean everything up. Turn off the arrayer and close arraymaker250.

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