MIAME platform type: spotted MIAME availability: Arrays printed at ISB can be purchased, contact bmarzolf@systemsbiology.org for information. Standard Operating Procedures for the Virtek(ESI) Spotter *** Always remember that spotter pins are extremely fragile at the tips *** To make spotter source plates: 1. Total volume should be 4-8 ul 2. Usually, DNA should be in 50% DMSO. This can be done most accurately be putting an appropriate amount of source material in spotting plates, then drying down with the speedvac and resuspending in the final volume of 3X SSC, 1.5M betaine. Things not to do: 1. Move slides after printing starts ? just touching a slide can shift it and other slides enough to ruin the grid geometry. 2. Use air to blow dust off of slides - you could move the slides 3. Press the RED EMERGENCY button if it's not a emergency- this will turn off the spotter controller and abort the entire run 4. Use PCR products that have been purified, then dried down and resuspended in a very small volume, this is known to cause erratic surface tension and cause spots to run together. When starting a new print run: 1. Allow blank slides to sit at 65% humidity for 24 hours 2. Renew sticky mat if it's dirty 3. Fill humidifier and turn on 4. Pull water out of water bath, wipe out bath and fill reservoir to get bath back to correct level- IT IS EXTREMELY IMPORTANT NOT TO OVERFILL THE WATERBATH OR SONICATOR BATH, WATER SHOULD BE JUST ABOVE THE POINT WHERE CUT RUNNING UP THE PIN ENDS 5. Place slides on the deck with label on the right-hand side. Secure slides by using spacer glass slides held in place with the magnets at the end of the platform. 6. Measure total X and Y distances spanned by slides, enter sizes under Calibration for Arraying->Slides 7. Print one dip with no source plate to ensure that pins are hitting all slides in the same plate (slight errors in Y direction especially can really throw off grid locations.) 8. Use the metal brace and spacer slides held by magnets to stabilize and line up the slides 9. Stabilize humidity at approximately 65% 10. Put blotting slide in place To print slides: 1. Spin down 4 source plates at 2000 rpm for one minute 2. Put three remaining source plates (in order) at 4 C 3. Check blotting slide for blocked pins, change slide. Remove clogged/sticking pins and try to remedy by cleaning pin and/or printhead hole. If a pin isn't spotting on the blotter, it may just not be contacting the blotter.see if it's been spotting on actual slides. 4. Change water bath water at beginning of run. 5. Check level of water reservoir and sonicator bath- add water if necessary 6. Check humidity 7. Double-check orientation and number of source plate 8. Press operator button 9. Re-seal old plate and put in cold room 10. Set timer 11. Check old plate out, new plate in on printing checklist When finished printing for the day: 1. Seal all plates and place in cold room 2. Turn off Spotter light 3. Turn monitor off 4. Leave computer ON When finished with entire printing: 1. Allow spotter humidity to slowly drop and slides dry out. 2. Remove slides and place into plastic Telechem slide boxes. 3. Turn off Spotter light 4. Turn off fan/humidifier 5. Place boxes in desiccator at least 12 hours. 6. Blow off slides in boxes, then bake at 85 C for 2 hours. 7. Etch numbers and corners on slides (corners on back side), blow dry and place in desiccator ready for prehyb. Things to do periodically: 1. Just keep an eye on the slides themselves to see if the grids are being formed as expected To clean a clogged pin: 1. Remove pin by pulling out from top with a small forceps. 2. Dip in water and blow dry with air tip, repeatedly if necessary. 3. Make sure water reservoir is sufficiently full - low water often causes clogging. Random experiments that we have tried and their results: 1. Effect of Temperature and Humidity on Spot Size Printed H-70 in 3X SSC onto Telechem Amine slides. Temperatures chosen were 18 C, 20 C, 22 C, and humidities of 40%, 50%, 60% resulting in 9 different conditions. Hybridization was with labeled PCR primer for 3 hours. Results: Differences were seen in specific grids, but grid-grid variability was too high to establish a conclusive pattern. Spots generally were most intense in a 90-100 micron diameter, then had a lighter halo that could extend to 130-150 microns. It appeared that Halo size was often smallest at 18 C, 130-140 microns. We decided to print at ambient temperature, generally 18-20 C. 2. Effect of Volume and Humidity on Maximum Spots per Dip Pooled 96 wells of purified PCR product and added SSC for a final concentration of 3X. Then set up a plate so that 8 pins would print 1ul, another 8 would print 2ul, 4ul, 6ul, 8ul and 10ul. This 48-pin dip was then printed 25 spots in the Y direction, across 10 plain glass slides for a total of 250 spots. This print was done with increasing indent at 75%, 70%, 65%, 60% and 55% humidity. Results: At 1-2ul, pins ran out of juice at 25-50 spots at all humidities. At 4ul, 200-225 spots could be printed at 65% or higher humidity. With 6ul or more, 250 spots could be printed at 65% or higher humidity. Based upon this, we chose 65% humidity for all printing.
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