Arichard:Protocols/MDA on microarray: Difference between revisions

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# Turn on microscope, stage, camera, and gas control. Manipulator and pipette pressure are not needed.
# Turn on microscope, stage, camera, and gas control. Manipulator and pipette pressure are not needed.
# Open valve on gas tank. Gauge should read ~20.
# Open valve on gas tank. Gauge should read ~20.
#
 
====Microscope program====
 
# Open multidimensional acquisition. Switch to BF filter with 1 ms exposure, 4X lens. Clear any previously saved positions.
# Pick three, non-overlapping positions for each square on the array. Save each position. Try to avoid picking fields of view near the edge.
# Ensure that the filter is now set to GFP, 200 ms exposure. CLOSE CURTAIN.
# 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.

Revision as of 20:30, 1 October 2012

MDA on microarray plate

  • DO NOT UV TREAT CELLS, NUCLEIC ACIDS, OR ENZYMES.
  • In general, everything else (reagents and tools) are UV treated prior to use.
  • 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 given in this protocol are per array.

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 litmus paper to confirm neutral pH.
  • All volumes given are exact and do not account for pipetting error. Adjust accordingly.
  1. Aliquot 1% BSA into 0.2 ul tube. Need 10 ul per array.
  2. Aliquot PBS into 0.2 ul tubes, 1 tube per array. Need 30 ul per array.
  3. Aliquot ALS into 0.2 ul tube. Need 4.5 ul per array.
  4. Aliquot NS into 0.2 ul tube. Need 4.5 ul per array.
  5. Aliquot MDA buffer into 0.2 ul tube. Need 2.2 ul per array.
  6. Aliquot mineral oil into 0.2 ul tubes, 1 tube per array. Need 60 ul per array.
  7. UV treat.
  8. Finish MDA master mix. Volumes given per array. This can be made during 5 min ALS incubation. Mix on ice block.
  • MDA master mix:
    • 2.2 ul buffer
    • 5 ul 200 uM N6
    • 0.8 ul 25 mM dNTP
    • 0.08 ul 50X SYBR Green
    • 1.6 ul phi29 Polymerase (Add last, immediately before use)

Plate prep

  • Each plate has 16 arrays, each of which is independent (separated by rubber gasket). Each array has ~150 wells.
  • Wells are 400 um ID, 100 um in height.
  • Do not transport the plate uncovered outside the hood.
  1. Plate must be ozone treated in Nano3 facility at Calit2 immediately prior to BSA coat. Ozone treatment lasts less than 1 hour.
  2. Wipe down hood, pipettes, and tip boxes with EtOH, then DNA away, then UV.
  3. Add 10 ul 1% BSA to each array. Be careful to coat entire array. Incubate 1 hour at RT.
  4. Wash 3x with 10 ul PBS. Leave the last wash on.
  5. Place plate in empty tip box for transport. Vacuum 30 seconds in vacuum centrifuge.

Protocol

Loading

  • All samples should be suspended at the correct dilution in 3 ul media (PBS, etc...).
  • Move tip back and forth across array while pipetting down, like a printer. Aim for uniform loading. Avoid the edges of the gasket.
  • Allow samples to settle 10 minutes before lysis.

Lysis

  • 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 ice block, 1 min off. Repeat 3x total.
  2. Add 4.5 ul ALS to each array, swirl to mix. Incubate 5 min at RT. Use this time to finish MDA master mix.
  3. Put on ice and add 4.5 ul NS, swirl to mix.
  4. Immediately add MDA master mix, swirl to mix.
  5. Overlay each array with 60 ul mineral oil. Ensure oil covers array. Bubbles are OK.

Microscope setup

  1. EtOH and DNA away all surfaces, then UV treat.
  2. In hood, place plate into gas box. Cover and tighten screws.
  3. Place gas box onto microscope stage and attach gas lines, left and right.
  4. Turn on microscope, stage, camera, and gas control. Manipulator and pipette pressure are not needed.
  5. Open valve on gas tank. Gauge should read ~20.

Microscope program

  1. Open multidimensional acquisition. Switch to BF filter with 1 ms exposure, 4X lens. Clear any previously saved positions.
  2. Pick three, non-overlapping positions for each square on the array. Save each position. Try to avoid picking fields of view near the edge.
  3. Ensure that the filter is now set to GFP, 200 ms exposure. CLOSE CURTAIN.
  4. Start acquisition BUT REMAIN INSIDE CURTAIN UNTIL FIRST ROUND OF IMAGES HAS BEEN ACQUIRED.
  5. After first time point acquisition, exit curtain and securely close with velcro strips.