Arichard:Protocols/MDA on microarray: Difference between revisions

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==MDA on microarray plate==
==MDA on microarray plate==


* DO NOT UV TREAT CELLS, NUCLEIC ACIDS, OR ENZYMES.
* Do not UV treat cells, nucleic acids, or enzymes.
* In general, everything else (reagents and tools) are UV treated prior to use.
* 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.
* UV treatment is 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.
* All volumes are per sample (array).
* All volumes exact; adjust accordingly for error.


===Prep===
===Prep===
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* ALS (Alkaline lysis solution) shelf life is two weeks.
* ALS (Alkaline lysis solution) shelf life is two weeks.
* To make ALS:
* To make ALS:
** 82 ul Nuclease free H2O
** 82 ul Nuclease free H2O.
** 8 ul 5 M KOH
** 8 ul 5 M KOH.
** 10 ul 1 M DTT
** 10 ul 1 M DTT.
** 2 ul 0.5 M EDTA
** 2 ul 0.5 M EDTA.
* KOH shelf life is also two weeks.
* KOH shelf life is also two weeks.
* 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.
* All volumes given are exact and do not account for pipetting error. Adjust accordingly.
* UV treat the following reagents:
# Aliquot 1% BSA into 0.2 ul tube. Need 10 ul per array.
** 1o ul 1% BSA per sample in a 0.2 ml tube.
# Aliquot PBS into 0.2 ul tubes, 1 tube per array. Need 30 ul per array.
** 30 ul PBS per sample in 0.2 ml tube, 1 tube per sample.
# Aliquot ALS into 0.2 ul tube. Need 4.5 ul per array.
** 4.5 ul ALS per sample in a 0.2 ml tube.
# Aliquot NS into 0.2 ul tube. Need 4.5 ul per array.
** 4.5 ul NS per sample in a 0.2 ml tube.
# Aliquot MDA buffer into 0.2 ul tube. Need 2.2 ul per array.
** 2.2 ul MDA buffer per sample in a 0.2 ml tube.
# Aliquot mineral oil into 0.2 ul tubes, 1 tube per array. Need 60 ul per array.
** 60 ul mineral oil in 0.2 ml tubes, 1 tube per sample.
# UV treat.
# 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====
====Plate prep====
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* Wells are 400 um ID, 100 um in height.
* Wells are 400 um ID, 100 um in height.
* Do not transport the plate uncovered outside the hood.
* 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 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.
# Wipe down hood, pipettes, and tip boxes with EtOH, then DNA away, then UV.
# Add 10 ul 1% BSA to each array. Be careful to coat entire array. Incubate 1 hour at RT.
# 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.
# Wash 3x with 10 ul PBS. Leave the last wash on.
# Place plate in empty tip box for transport. Vacuum 30 seconds in vacuum centrifuge.
# Vacuum 30 seconds in vacuum centrifuge.
# Remove last wash. Vacuum again 30 seconds.
# UV treat 10 min.


===Protocol===
===Protocol===
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* All samples should be suspended at the correct dilution in 3 ul media (PBS, etc...).
* All samples should be suspended at the correct dilution in 3 ul media (PBS, etc...).
* Each sample will be loaded 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.
* 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.
* Allow samples to settle 10 minutes before lysis.
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# 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.
## 1 minute on ice block, 1 min off. Repeat 3x total.
## 1 minute on dry ice, 1 min off. Repeat 5x total.
# Add 4.5 ul ALS to each array, swirl to mix. Incubate 5 min at RT. Use this time to finish MDA master mix.
# 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:
## 2.2 ul MDA 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).
# Put on ice and add 4.5 ul NS, swirl to mix.
# Put on ice and add 4.5 ul NS, swirl to mix.
# Immediately add MDA master mix, swirl to mix.
# Immediately add 9.68 ul MDA master mix, swirl to mix.
# Overlay each array with 60 ul mineral oil. Ensure oil covers array. Bubbles are OK.
# Overlay each array with 60 ul mineral oil. Ensure oil covers array. Bubbles are OK.


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# Place gas box onto microscope stage and attach gas lines, left and right.
# Place gas box onto microscope stage and attach gas lines, left and right.
# 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 UNITS NEEDED.
#
 
====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.

Latest revision as of 01:27, 27 November 2012

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 is 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 litmus paper to confirm neutral pH.
  • UV treat the following reagents:
    • 1o 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.
    • 2.2 ul MDA buffer per sample in a 0.2 ml tube.
    • 60 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 ~150 wells.
  • Wells are 400 um ID, 100 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.

Protocol[edit]

Loading[edit]

  • All samples should be suspended at the correct dilution in 3 ul media (PBS, etc...).
  • Each sample will be loaded 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.
  • Allow samples to settle 10 minutes before lysis.

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. 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. 2.2 ul MDA buffer.
    2. 5 ul 200 uM N6.
    3. 0.8 ul 25 mM dNTP.
    4. 0.08 ul 50X SYBR Green.
    5. 1.6 ul phi29 Polymerase (Add last, immediately before use).
  3. Put on ice and add 4.5 ul NS, swirl to mix.
  4. Immediately add 9.68 ul MDA master mix, swirl to mix.
  5. Overlay each array with 60 ul mineral oil. Ensure oil covers array. Bubbles are OK.

Microscope setup[edit]

  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 UNITS NEEDED.

Microscope program[edit]

  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.