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.
- Aliquot 1% BSA into 0.2 ul tube. Need 10 ul per array.
- Aliquot PBS into 0.2 ul tubes, 1 tube per array. Need 30 ul per array.
- Aliquot ALS into 0.2 ul tube. Need 4.5 ul per array.
- Aliquot NS into 0.2 ul tube. Need 4.5 ul per array.
- Aliquot MDA buffer into 0.2 ul tube. Need 2.2 ul per array.
- Aliquot mineral oil into 0.2 ul tubes, 1 tube per array. Need 60 ul per array.
- 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
- 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.
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.
- 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.
- 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.
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.