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Arichard:Protocols/MDA on microarray 2013 09 05
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==MDA on microarray plate== * Do not UV treat cells, nucleic acids, or enzymes. * UV treat all other reagents, as well as tools (e.g., pipettes and tips). * UV treatment means 10-15 minutes under UV. All tubes must be open as plastic shields from UV. Smaller volumes are treated more efficiently. * All volumes are per sample (array). * All volumes exact; adjust accordingly for error. ===Prep=== * Work in the hood. * Use fresh tips every time. * Use tweezers to remove tubes from containers. ====Reagent prep==== * PBS is syringe filtered through 0.2 um into 50 ml tube. * ALS (Alkaline lysis solution) shelf life is two weeks. * To make ALS: ** 82 ul Nuclease free H2O. ** 8 ul 5 M KOH. ** 10 ul 1 M DTT. ** 2 ul 0.5 M EDTA. * KOH shelf life is also two weeks. * NS is neutralization solution. * Check the pH by mixing 2 ul ALS with 2 ul NS. Mix well by pipette and drop onto Whatman paper to confirm neutral pH. * The following reagents should be UV treated prior to use: ** 10 ul 1% BSA per sample in a 0.2 ml tube. ** 30 ul PBS per sample in 0.2 ml tube, 1 tube per sample. ** 4.5 ul ALS per sample in a 0.2 ml tube. ** 4.5 ul NS per sample in a 0.2 ml tube. ** 2.3 ul MDA buffer and 5 ul H2O per sample in a 0.2 ml tube. ** 80 ul mineral oil in 0.2 ml tubes, 1 tube per sample. ====Plate prep==== * Each plate has 16 arrays, each of which is independent (separated by rubber gasket). Each array has 247 wells. * Wells are 400 um ID, 160 um in height. * Do not transport the plate uncovered outside the hood. # Plate must be O2 plasma treated in Nano3 facility at Calit2 immediately prior to BSA coat. Treatment lasts less than 1 hour. # Wipe down hood, pipettes, and tip boxes with EtOH, then DNA away, then UV. # UV treat the plate after plasma treatment. # Add 10 ul 1% BSA to each array. Be careful to coat entire array. Vacuum 30 seconds in vacuum centrifuge, then UV treat and let sit 1 hour at RT. # Wash 3x with 10 ul PBS. Leave the last wash on. # Vacuum 30 seconds in vacuum centrifuge. # Remove last wash. Vacuum again 30 seconds. # UV treat 10 min. Also wipe down stage adapter, cover, and metal thermocycler adapter (for freeze thaw) with EtOH, DNA away, and UV treat. ===Protocol=== ====Loading==== * All samples should be suspended at the correct dilution in 3 ul media. ** Samples should also be stained at 1X with SYBR Green if the array loading is to be imaged prior to lysis. * Load each sample into its own array. Move tip back and forth across array while pipetting down, like a printer. Aim for uniform loading. Avoid the edges of the gasket. * Cover with coverslip and press down to seal against gasket. * To image array loading: Image under fluorescence (GFP channel), saving each image and position using multi-dimensional acquisition. Record 3 non-overlapping images per array. Cells will also settle during this time, ~10 min. ** Imaging is not necessary for chromosomal samples, as loading concentration will be high enough to assume every well contains template. Let chromosomal samples settle for 10 min. ====Lysis==== * Mixing is important for uniform array performance. Avoid touching rubber gasket with pipette tip, however. # In order to lyse bacteria, plate must be freeze thawed. This is only necessary for bacteria, but will not harm eukaryotic samples. ## Cover with cover slip. ## 1 minute on dry ice, 1 min off on thermocycle adapter. Repeat 5x total. # Add 4.5 ul ALS to each array, swirl to mix. Incubate 10 min on ice block. Use this time to finish MDA master mix: ## 2.3 ul MDA buffer. ## 1 ul 1 mM *N6. ## 1 ul 25 mM dNTP. ## 0.1 ul 50X SYBR Green. ## 1.8 ul phi29 Polymerase (Add last, immediately before use). ## 5 ul H2O # Put on ice and add 4.5 ul NS, swirl to mix. # Immediately add 11.2 ul MDA master mix, swirl to mix. # Overlay each array with 75 ul mineral oil. Ensure oil covers array. Bubbles are OK. ====Microscope setup==== # EtOH and DNA away all surfaces, including stage adapter and cover. UV treat. # In hood, place plate into stage adapter. Cover and tighten screws. # Place stage adapter onto microscope stage. # Turn on microscope, stage, camera, and temperature control. Manipulator and pipette pressure are not needed. ====Microscope program==== # Ensure that the filter is now set to GFP, 50 ms exposure. # Set acquisition for 11 timepoints, 60 min intervals. # Close curtain and start acquisition, but remain inside curtain until first round of images has been acquired. # After first time point acquisition, exit curtain and securely close with velcro strips.
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