Arichard:Protocols/MDA on microarray: Difference between revisions

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* In general, everything else (reagents and tools) are UV treated prior to use.
* 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.
* 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===
====Reagent prep====
* PBS is syringe filtered through 0.2 um into 50 ml tube.
* PBS is syringe filtered through 0.2 um into 50 ml tube.
* ALS (Alkaline lysis solution) shelf life is two weeks.
* ALS (Alkaline lysis solution) shelf life is two weeks.
Line 14: Line 17:
* NS is neutralization solution.
* 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.
* 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.
* 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.
* Volumes in protocol are given per array.
* Mixing is important for uniform array performance. Avoid touching rubber gasket with pipette tip, however.
* Do not transport the plate uncovered outside the hood.
===Prep===
====Reagent prep====
* All volumes given are exact and do not account for pipetting error. Adjust accordingly.
* All volumes given are exact and do not account for pipetting error. Adjust accordingly.
# Aliquot 1% BSA into 0.2 ul tube. Need 10 ul per array.
# Aliquot 1% BSA into 0.2 ul tube. Need 10 ul per array.
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** 1.6 ul phi29 Polymerase (Add last, immediately before use)
** 1.6 ul phi29 Polymerase (Add last, immediately before use)
====Plate prep====
====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.
# Plate must be ozone treated in Nano3 facility at Calit2 immediately prior to BSA coat. Ozone treatment lasts less than 1 hour.
# Plate must be ozone treated in Nano3 facility at Calit2 immediately prior to BSA coat. Ozone treatment lasts less than 1 hour.
# Wipe down hood, pipettes, and tip boxes with EtOH, then DNA away, then UV.
# Wipe down hood, pipettes, and tip boxes with EtOH, then DNA away, then UV.
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* Allow samples to settle 10 minutes before lysis.
* Allow samples to settle 10 minutes before lysis.
====Lysis====
====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.
# 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.
## Cover with cover slip.

Revision as of 23:45, 14 September 2012

MDA on microarray plate

  • Work in the hood.
  • Use fresh tips every time.
  • 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

Reagent prep

  • PBS is syringe filtered through 0.2 um into 50 ml tube.
  • ALS (Alkaline lysis solution) shelf life is two weeks.
    • 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.
    • 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.