WHRobinson Lab, Stanford University School of Medicine
Manufacture protocol
Making the arrays: 1. Cut one sandwich of PVDF membrane into six equal-sized pieces. PVDF Membrane Filter Paper Sandwich, (0.2 uL Pore Zie, #LC2002, Invitrogen). Trim the vertical edges of the membrane first to eliminate excess cover paper; 2. Using two pieces of double-sided 3M Scotch tape, tape each membrane onto the middle of a pre-cleaned microscope slide (Gold Seal Microslides, Cat#3050). Avoid tape overlap and ensure that tape meets the edge of the membrane; 3. Align the membrane using an old slide as a guide; 4. Trim extra membrane or tape portions off the edge of the slide.
Printing the arrays: CAMAG Automatic TLC Sampler 4 (ATS4) robot. 1. Turn on the nitrogen gas by turning the large silver knob to the left. Gas is usually set to 65-75 psi, so other knobs should not have to be adjusted; 2. Turn on arrayer by pushing the button on the back left-hand side of the machine. Arrayer display should read “ATS4 System Ready.”; 3. Check the “Rinse” (right, methanol) and “Waste” (left) bottles. Refill or empty as necessary; 4. Rinse the system by hitting “End” (display will read “this job clean rinse unit”), and then “Enter.”; 5. Obtain and organize 0.01 ml lipid aliquots from cold room and 4C fridge; 6. Mark slides using pencil with the array number and the date of the print. Check the arrays for cracks in the glass, and to make sure the PVD membrane goes to the edge of the slide and line up with membranes from the other slides; 7. Place each slide in appropriate slot on arrayer. Push down on the slide to make sure it is secured in place. For slides that are loose, push towards lower left-hand corner of the slot. Once all slides are in place, run finger down the end of each to secure again; 8. Push platform up (back) and left, and lock with lever on right-hand side; 9. For each aliquot, remove all black caps with decrimper and push on the plastic orange caps; 10. Place aliquots in machine, giving each a slight shake to make sure they are thawed; 11. Close lid of arrayer; 12. On Desktop, Open “Wincats.”; 13. Enter Login ID and password; 14. Under Instruments, select ATS4 “Freemode”; 15. Select File, then Open “8x11x12partA50” **The Arrayer uses two program files to spot each slide: 8x11x12partA50 (lipids 1-25) and 8x11x12partB50 (lipids 26-50). The Arrayer will spot program A on all twelve slides in each run and then spot program B; 16. Under Dosage, select Start. The arrayer will begin printing; 17. Watch arrayer carefully to note any problems. Once Program A is complete, the arrayer will make a sound and the screen backdrop will turn blue. Switch to Program B at this time (Select File, then Open “8x11x12partB50” then select Dosage, “Start”); 18. After each print run (every 12 slides), check aliquot levels and substitute fresh aliquots if need be. Repeat Steps 6-11, and 15-17 for each run; 19. At the end of the printing, being a Rinse cycle (hit “End”); 20. Turn off machine and store printed arrays appropriately.
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