Whatman FAST slides, proteins spotted in Silverman Lab, UCSD School of Medicine
Manufacture protocol
Microarray Printing Protocol 1. Load Q soft microarray program/ turn on the microarray machine- do not turn it off while the program is on. 2. Load the 2005916 dual profile. 3. Click on "change head" icon. 4. Remove the pins once the machine moves the printing head. 5. Wash the printing pins using aQuclean diluted to 2% solution with DI water. Place the pins on four corners of pin container. Pour 2% aQuclean solution into the sonicator then submerge the printing head into the sonicator for 15 minutes then 5 minutes in DI water with the sonicator on. It is recommended to "de-gas" without the printing head in the sonicator- run the sonicator with 2% aQuclean without the printing head. (Sonicator runs for 5 minutes per cycle, therefore you have to run the cycle 3 times) 6. Flush the tube every once in a while. 7. Water and ethanol tanks fit 5L. Fill the water tank with nano pure water from the back room. Turn on the filter by pressing the green button on top left. "17" or better is adequate for use. For ethanol(not actualy ethanol but tween solution), clean out the tank with DI water first then measure 4L nano pure water in cylinder. Pour the nano pure water into a plastic tank. Use serological pipette to measure 2mL tween (Tween BP276500 Fischer), place it in plastic tank with the 4L nano pure water. 8. Drain out waste tank before running the microarray machine. 9. In room 120, check the clean printing pins under microscope for dirt, or anything that might be lodged in between the small space at the tip of the pins. - Clean the glass with kimwipe using 70% EtOH. - Clean the pins with kimwipe then wipe excess moisture. - Keep the orientation of the pins constant at all times- this may allow us to pinpoint the problematic pin in printing error. 10. Dump out aQuclean solution in the sonicator then rinse with DI water. Fill the sonicator with nano pure water and let the sonicator run for a while without the printing pins in it. Run the sonicator with the printing tips for 5 minutes. 11. Clean and wipe the machine with 70% EtOH using kimwipes. Wipe in between the panels, back of the panel, etc. Also use the ethanol wash bottle to clean the pins. 12. Place 3 epoxy slides on bottom right most corner for "pre-printing." Place 50 Whatman slides (item# 10485317) on the panel, ensuring that the slides are touching the top & right corners of each printing panels. Cover the last printing panel as all printing panels must be covered in order for the vacuum to work. Keep same lot # for all the slides printed together. Close the lid. 13. Find sample plates from the refrigerator with "REVCO" sticker. Centrifuge the array sample for compression. Balance the sample plate using a plate that is of same weight. (can be found in cabinet next to the GS-6R centrifuge) Use two green weight to balance the plates. Centrifuge at speed ~1800 for a minute or so. 14. After the centrifuge, peel off the aluminum from the sample plate then place the sample plate in Platform right next to the raised black wash station on the right-hand side of the miroarray machine. Turn on the machine, ensure that the vacuum is on auto. Pour nano pure water into nebulizer, then tightly seal the opening. 15. Open Qsoft microarraying program. Loading 2005916 Dual. Turn on chiller and vacuum, then humidity, using icons in the program. 16. Place the pins in the right most 4 corners. Click on "run"- type: normal, no change in head-yes: Go ahead 17. Fill out the information on slide batches- mark date and slide lot # and list slide barcode #s. 18. The machine runs for ~ 5 hours. GAL File Generation Protocol 3/3/08 (see attached example file for further clarification): 1. Open data tracking template in Excel a. Input Names of Antigens b. Input ZZZ for barcode, accession number, and comments for column 2, 4 and 6 c. Assign Unique name to library name, column 1 2. Save as CSV (comma-separated values) format in excel 3. Open CSV file in Notepad and copy all 4. Re-open excel and paste. All data will now be on one column a. Save as .txt (Tab-delimited) file b. Reopen file in notepad and make sure that the format is as below: i. "20060801_hu array_20061002,zzz,PLATE LOCATION,zzz,SAMPLE NAME ,zzz" ii. Make sure that there are NO QUOTES in the first four lines iii. Make sure that there are no commas within any given field for the rest of the lines, otherwise you will have to manually find this and delete the commas later 5. Copy .txt file onto Qarray laptop 6. Open QArray DataTracking 7. Tools->Import Process File -> Our .txt file a. Next, next, finish… 8. Open QArray Mini, Load the current routine from which the slides were printed 9. Data Tracking Tab a. Make sure that the export data check mark is checked b. Set a file name for your gal file c. File format: Gal 4.1 or Gal d. Double click groups i. Double click the current routine within groups ii. Click the Library Manager import entry iii. Add the entry that we have made earlier, in step 7 iv. Delete any other entries within this group 10. Go to Start Tab a. Run Type -> Data Tracking Export Only b. Run c. The .xml file will be saved in the same directory as your .txt file that you loaded in step 7 d. The .gal file will be saved in “C:/Program Files/Genetix/QArray/Logs/”, as the filename you gave it 11. Transfer this file to the scanner computer or the other Dell desktop to perform Spot ID protocol.
