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. | ||
* | * UV treat all other reagents, as well as tools (e.g., pipettes and tips). | ||
* UV treatment | * 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 | * All volumes are per sample (array). | ||
* All volumes exact; adjust accordingly for error. | |||
===Prep=== | ===Prep=== | ||
Line 17: | Line 18: | ||
* 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. | ||
* | * 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==== | ====Plate prep==== | ||
Line 50: | Line 43: | ||
# 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. | # Place plate in empty tip box for transport. Vacuum 30 seconds in vacuum centrifuge. | ||
# Remove last wash. Vacuum again 30 seconds. | |||
===Protocol=== | ===Protocol=== | ||
Line 56: | Line 50: | ||
* 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. | ||
Line 65: | Line 60: | ||
## Cover with cover slip. | ## Cover with cover slip. | ||
## 1 minute on ice block, 1 min off. Repeat 3x total. | ## 1 minute on ice block, 1 min off. Repeat 3x 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 5 min at RT. 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 MDA master mix, swirl to mix. | ||
Line 76: | Line 76: | ||
# 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==== | ====Microscope program==== | ||
Line 82: | Line 82: | ||
# Open multidimensional acquisition. Switch to BF filter with 1 ms exposure, 4X lens. Clear any previously saved positions. | # 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. | # 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. | # Ensure that the filter is now set to GFP, 200 ms exposure. Close curtain. | ||
# Start acquisition | # 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. | # After first time point acquisition, exit curtain and securely close with velcro strips. |
Revision as of 18:17, 12 October 2012
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 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
- 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.
- 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
- 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.
- 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.
- 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.
- Remove last wash. Vacuum again 30 seconds.
Protocol
Loading
- 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
- 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 ice block, 1 min off. Repeat 3x 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:
- 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.
- Immediately add MDA master mix, swirl to mix.
- Overlay each array with 60 ul mineral oil. Ensure oil covers array. Bubbles are OK.
Microscope setup
- EtOH and DNA away all surfaces, then UV treat.
- In hood, place plate into gas box. Cover and tighten screws.
- 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.
- 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.