Arichard:Protocols/MDA on microarray 2013 09 05
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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 means 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 Whatman paper to confirm neutral pH.
- The following reagents should be UV treated prior to use:
- 10 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.
- 2.3 ul MDA buffer and 5 ul H2O per sample in a 0.2 ml tube.
- 80 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 247 wells.
- Wells are 400 um ID, 160 um in height.
- Do not transport the plate uncovered outside the hood.
- 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.
- 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.
- Vacuum 30 seconds in vacuum centrifuge.
- Remove last wash. Vacuum again 30 seconds.
- UV treat 10 min. Also wipe down stage adapter, cover, and metal thermocycler adapter (for freeze thaw) with EtOH, DNA away, and UV treat.
Protocol[edit]
Loading[edit]
- All samples should be suspended at the correct dilution in 3 ul media.
- Samples should also be stained at 1X with SYBR Green if the array loading is to be imaged prior to lysis.
- Load each sample 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.
- Cover with coverslip and press down to seal against gasket.
- To image array loading: Image under fluorescence (GFP channel), saving each image and position using multi-dimensional acquisition. Record 3 non-overlapping images per array. Cells will also settle during this time, ~10 min.
- Imaging is not necessary for chromosomal samples, as loading concentration will be high enough to assume every well contains template. Let chromosomal samples settle for 10 min.
Lysis[edit]
- 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 dry ice, 1 min off on thermocycle adapter. Repeat 5x total.
- 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:
- 2.3 ul MDA buffer.
- 1 ul 1 mM *N6.
- 1 ul 25 mM dNTP.
- 0.1 ul 50X SYBR Green.
- 1.8 ul phi29 Polymerase (Add last, immediately before use).
- 5 ul H2O
- Put on ice and add 4.5 ul NS, swirl to mix.
- Immediately add 11.2 ul MDA master mix, swirl to mix.
- Overlay each array with 75 ul mineral oil. Ensure oil covers array. Bubbles are OK.
Microscope setup[edit]
- EtOH and DNA away all surfaces, including stage adapter and cover. UV treat.
- In hood, place plate into stage adapter. Cover and tighten screws.
- Place stage adapter onto microscope stage.
- Turn on microscope, stage, camera, and temperature control. Manipulator and pipette pressure are not needed.
Microscope program[edit]
- Ensure that the filter is now set to GFP, 50 ms exposure.
- Set acquisition for 11 timepoints, 60 min intervals.
- Close curtain and 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.