Arichard:Protocols/MDA amplicon extraction and processing 2013 09 05
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MDA amplicon extraction and processing[edit]
- Volumes given are exact and given per reaction (each extracted sample). Adjust accordingly for error.
- Use low-binding tubes and tips when handling DNA.
Prep[edit]
- Setup the following before checking for amplification. UV treat:
- 1 ul H2O in each 0.2 ml tube (low-bind), 1 tube per sample.
- ~20 glass micropipets, in UV oven.
- The following reagents will be needed immediately following extraction. UV treat:
- 1.5 ul ALS per rxn in 0.2 ml tube.
- 1.5 ul NS per rxn in 0.2 ml tube.
- Pol I master mix (per rxn).
- 1 ul 10X NEB buffer 2.
- 11.6 ul H2O.
- Prepare the EtOH precipitation mix during Pol I treatment. UV treat in 1.5 ml tube, 1 tube per sample:
- 50 ul 100% EtOH
- 2 ul 3 M NaAc
- 1 ul glycoblue.
- During EtOH precipitation, UV treat:
- 7 ul H2O per rxn for resuspending.
- 2 ul HMW (or LMW for prokaryotic samples) per rxn, if proceeding directly to tagmentation.
Protocol[edit]
Amplicon extraction[edit]
- A trained eye is required to detect amplification without processing image to remove background.
- A "digital" amplification profile is expected, which is to say a poissonian distribution of positive wells, with ~10% of wells showing amplification and no local clusters of positive wells. Essentially, we expect ~25 positive wells out of each array of 247, evenly scattered over the array (except perhaps around the edges).
- Amplification usually begins with a green spot at the edge of the well observable at timepoint 1, with subsequent expansion to either fill the well or form a crescent centered at the point of the original spot.
- Bubbles autofluoresce. Bubbles can be identified by 3 criteria:
- Movement between timepoints.
- Located in between wells or over dead space between wells.
- Uniform intensity over time (no amplification).
- We typically monitor 3 non-overlapping positions for each reaction. The number of replicates may vary. Not every position may be needed to extract enough replicates. Every well is assumed to be independent (this cannot be rigorously confirmed until sequencing).
- Loosen red screws and remove cover from stage adapter.
- Turn on pressure control and manipulator.
- Open multiple time point images for a given position (e.g., first, middle, and last). Identify a positive well using the criteria discussed above.
- Mount pipette tip. Carefully swing manipulator arm into position and tighten. It may be necessary to twist pipette up in order to clear the ledge, then twist back down and tighten over the plate.
- Using BF filter, 1 ms exposure, 4x lens, find the target well.
- Remove the oil overlay and any excess reaction mix using a P200. Pipet from the edges of the array. Only remove the oil from one array at a time to avoid evaporation.
- Position the pipette over the well. Begin to lower the pipette in the z-axis. Do not go too fast, as the controller will take this as a queue to switch resolution (it starts moving really fast, stabs the plate).
- There will be a change in refraction as the pipette enters the reaction mix. At this point the pipette should be in the well. Move the tip to the edge of the well and observe surrounding wells to assess for deformation, indicating that the tip is in contact with the well wall and fully inside the well.
- Apply only as little negative gauge pressure as is necessary to extract the buffer and amplicons. This will vary for different tip diameters and geometries. Extraction must be assessed by eye.
- Zero out the gauge pressure and home the manipulator. Loosen the manipulator screw and carefully swing the arm out (again, take care not to impact on the lip of the box).
- Immerse the tip in the 1 ul drop of H2O in a 0.2 ul tube. Apply positive pressure, then clear. Drag the tip along the side of the tube to remove any excess droplets that may be retained.
- Cap tube for transport to hood.
Amplicon processing[edit]
- Briefly spin samples to collect fluid.
- Add 1.5 ul ALS, incubate 3 minutes at RT. Mix well by pipette.
- Complete the Pol I master mix (per rxn):
- 0.4 ul 25 mM dNTP
- 1 ul 370 ng/ul N6
- 1 ul 10X AMP ligase buffer
- 1 ul Pol I
- Move reaction to ice block and add 1.5 ul NS. Mix well by pipette.
- Add 16 ul DNA Pol I master mix to each tube. Mix well by pipette and incubate 60 minutes at 37 degC, followed by 10 minutes at 65 degC.
- Add 0.2 ul AMP ligase. Mix well by pipette and incubate 10 min at 37 degC followed by 10 min at 65 degC.
- Add reaction to EtOH precipitation mix. Incubate 20 minutes at -80 degC.
- Spin 25 minutes at 10,000 RPM (10,600 RCF) at 4 degC.
- Carefully remove and discard supernatant and add 750 ul of 75% EtOH to each pellet.
- Spin 5 minutes at 10,000 RPM at 4 degC.
- Carefully remove and discard supernatant and let dry 5 minutes.
- Add 7 ul H2O to each pellet and resuspend by pipetting. Store at 4 degC or proceed directly to tagmentation and amplification.