Arichard:Protocols/MDA on microarray 2013 09 05

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MDA on microarray plate[edit]

  • 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[edit]

  • Work in the hood.
  • Use fresh tips every time.
  • Use tweezers to remove tubes from containers.

Reagent prep[edit]

  • 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[edit]

  • 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.
  1. Plate must be O2 plasma treated in Nano3 facility at Calit2 immediately prior to BSA coat. Treatment lasts less than 1 hour.
  2. Wipe down hood, pipettes, and tip boxes with EtOH, then DNA away, then UV.
  3. UV treat the plate after plasma treatment.
  4. 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.
  5. Wash 3x with 10 ul PBS. Leave the last wash on.
  6. Vacuum 30 seconds in vacuum centrifuge.
  7. Remove last wash. Vacuum again 30 seconds.
  8. 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[edit]

Loading[edit]

  • 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[edit]

  • Mixing is important for uniform array performance. Avoid touching rubber gasket with pipette tip, however.
  1. In order to lyse bacteria, plate must be freeze thawed. This is only necessary for bacteria, but will not harm eukaryotic samples.
    1. Cover with cover slip.
    2. 1 minute on dry ice, 1 min off on thermocycle adapter. Repeat 5x total.
  2. 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:
    1. 2.3 ul MDA buffer.
    2. 1 ul 1 mM *N6.
    3. 1 ul 25 mM dNTP.
    4. 0.1 ul 50X SYBR Green.
    5. 1.8 ul phi29 Polymerase (Add last, immediately before use).
    6. 5 ul H2O
  3. Put on ice and add 4.5 ul NS, swirl to mix.
  4. Immediately add 11.2 ul MDA master mix, swirl to mix.
  5. Overlay each array with 75 ul mineral oil. Ensure oil covers array. Bubbles are OK.

Microscope setup[edit]

  1. EtOH and DNA away all surfaces, including stage adapter and cover. UV treat.
  2. In hood, place plate into stage adapter. Cover and tighten screws.
  3. Place stage adapter onto microscope stage.
  4. Turn on microscope, stage, camera, and temperature control. Manipulator and pipette pressure are not needed.

Microscope program[edit]

  1. Ensure that the filter is now set to GFP, 50 ms exposure.
  2. Set acquisition for 11 timepoints, 60 min intervals.
  3. Close curtain and start acquisition, but remain inside curtain until first round of images has been acquired.
  4. After first time point acquisition, exit curtain and securely close with velcro strips.