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Please ensure that there is no EDTA in your input DNA elution buffer. We’ve experienced over-tagmentation when EDTA is present in the tagmentation reaction. Eluting your input DNA in RSB, Tris-HCl pH 8.0, or water are all suitable.

Data from samples sheared outside of our recommendation cannot be guaranteed. Smaller fragments will likely be discarded during bead clean-ups due to stringent ratios. While larger fragments will likely be retained, the final library concentration (in nM) is expected to decrease.

At Miroculus we shear DNA in 50ul using the microTUBE AFA Fiber Screw-Cap 6x16mm (Covaris PN 520096) and a Covaris M220 instrument.
We have sheared 500ng to 2250ng of DNA per 50ul using the following settings:

Peak Power: 50
Duty Factor: 20
Cycle/Burst: 200

The shearing duration varies from 80sec to 140sec and has to be optimized for each new batch of gDNA. The target shearing size is to have a peak distribution between 360bp and 450bp.

For customers using an LE220 Covaris instrument with single tubes or 8-strips:

Peak Incident Power: 450
Duty Factor: 15-30
Cycles/Burst: 200
Shear Time: 60-100 sec

Yes, please adjust the volume of 15 µM adapters from 3.6 µl to 5 µl if you are using 1 µg of input DNA.

Final library yields can be impacted by input sample quality. We recommend examining your input DNA using a Bioanalyzer® or similar instrument.

Additionally, library yields can be affected by adapters that have undergone too many freeze-thaw cycles. We recommend aliquoting adapters to avoid this.

Finally, we recommend that the 80% ethanol solution used in protocol purification steps is made fresh. If not, this solution could be less than 80% ethanol (due to evaporation over time) and therefore causing inefficient purification of DNA throughout the workflow.

In the instrument, HV is required for droplet mobility. The HV indicator light, located at the top right of the electrode board will illuminate yellow when HV is functioning normally. HV will not turn on if the cartridge clamp is engaged without a cartridge loaded in the instrument.

The instrument contains four magnet zones, in which magnets can be independently engaged or disengaged during bead-based purification steps within the protocol.

The instrument contains heaters for both thermocycling and isothermal temperature control, which are used for reagent incubation steps.

During a run, the instrument is able to detect the presence, location, and approximate size of reagent droplets, but not Dropgloss.

When the touchscreen indicates that the protocol has ended and it is time to recover the sample, lift the lid by securing the instrument base with one hand, grasping the center of the lid with your other hand and lifting straight up. Do not tip the instrument, as this may move your sample away from the recovery inlet. Be sure to leave the cartridge clamp engaged during sample recovery. To recover your sample, set a 200 μl pipette 20 μl higher than the volume of the sample you are recovering, depress the plunger to the first stop, insert the tip vertically into the recovery inlet, and slowly release the plunger to draw up your sample. Transfer the sample to a clean, labeled tube for analysis and storage.

When indicated on the touchscreen, load the prepared Reservoir Reagents into reservoirs.

Remove the blue cap from the reservoir. To load, draw up the reagent using a 1000 µl pipette fitted with a barrier tip. Hold the pipette vertically and fully insert until met with resistance to dispense. Remove the tip from the reservoir before releasing the pipette plunger. To seal the reservoir, fully press the blue cap onto the reservoir. Reagents loaded into reservoirs cannot be automatically detected as inlet reagents can. Press Continue on the touchscreen to advance to the next reservoir.

Dropgloss will solidify below 18C. If your Dropgloss has solidified in the storage bottle, hold the bottle in your hand or place it in a 25C chamber until it goes back into solution. Vortex well before use. Store Dropgloss on the benchtop in an area that is consistently above 18C. While some stock reagents should be kept on ice, reaction mixes should be kept at room temperature on the benchtop until you are ready to load the cartridge. Do NOT keep reaction mixes on ice.
If your Dropgloss has solidified in your reaction mix, hold the tube in between your fingers until it melts, then briefly vortex and pulse-spin to collect contents. Store at ambient temperature until ready to load into the Miro Cartridge.

Regurgitation when loading cartridge inlets (potentially due to a pipette tip that is too wide, held at too severe of an angle, or not inserted fully) results in droplet splitting that cannot be rescued. The safest response is to discard your current cartridge and load a fresh cartridge. Restart the run. Any attempts to proceed with or without adding back what was lost may compromise the quality of the final library.

If after a minute the reagent remains undetected, high voltage (HV) may not be functioning as expected, potentially due to the clamp not being closed in a timely manner. If the HV indicator light is off, attempt to recover the reagent from the inlet, restart the run and ensure that HV is functioning (as indicated by a yellow indicator light), and re-load the cartridge.

Alternatively, the reagent may be loaded into the wrong inlet. Attempt to recover the reagent from the inlet and re-load into the correct inlet. Finally, it may be that only Dropgloss was loaded into the inlet. Attempt to recover the Dropgloss from the inlet and re-load reagent+Dropgloss.

Dropgloss is viscous, and as a result, some residual amount can cling to the wall of the tubes/tips. This has been accounted for in the protocol and it is expected to have a low amount of Dropgloss remaining in the tube/tip after removing the reagents to add to the cartridge. To maximize recovery, pulse spin the tubes before pipetting. Follow pipetting best practices, hold the pipette tip vertically in the tube and draw liquid up from the bottom of the PCR tube slowly.

There are two types of bubbles that can be seen in the pipette tip during reagent loading into the inlets. The first is air bubbles. Extreme care should be taken to avoid having air bubbles within the reagent in the pipette tip as they can inhibit mobility of the droplet, or pop during movement which could separate the droplets. The second is an ‘oil in vinegar’ effect, which arises from mixing the aqueous reagents with the Dropgloss. These oil in vinegar bubbles will not negatively impact the run, however if the customer is unclear about whether it is air or oil, they should try to let them resolve before loading. They can just wait a few seconds or gently tap the pipette to aid in letting them resolve.

When indicated on the touchscreen, load the prepared reagents into Miro Cartridge inlets.

First, pulse spin the reagents to bring all contents to the bottom of the tube. Next, set a 200 μl pipette* to 20 μl greater than the collective volume of the reagent and Dropgloss, and draw liquid up from the bottom of the PCR tube**. Drawing up into the pipette tip in this way ensures that any small volume of liquid left in the PCR tube is Dropgloss, which will not negatively affect the run. If you notice bubbles in the pipette tip, wait for the bubbles to resolve before dispensing into the inlet.

To load, fully insert the tip into the inlet, then depress the plunger in a single, swift, and smooth motion until you reach the first stop of the pipette.*** Hold the depressed pipette in the inlet for 3 seconds. Remove the pipette tip from the inlet before releasing the plunger. It may take a moment for the reagent to be detected once loaded into the cartridge inlet. Once detected, the touchscreen will automatically prompt you to load the next inlet reagent.

*Use a 200 μl pipette when loading inlets, as a smaller pipette may puncture or deform the cartridge film and a larger pipette will not fit appropriately in the inlet. 

**For reaction mixes containing >50 μl Dropgloss, place the pipette tip in the top layer (Dropgloss) of liquid and draw up as much as possible, then lower the tip to the bottom of the tube and draw up the remaining liquid from the bottom.

***Air in the pointed end of the pipette tip is normal and will harmlessly dissipate into the air within the cartridge. However, depressing the pipette past the first stop can introduce an air bubble into the reagent droplet, which can negatively affect the run.

Standard 200 μl barrier pipette tips are best, as smaller pipette tips, when inserted fully, will touch and could puncture the film on the bottom of the cartridge. Any resulting film deformations can negatively affect the run, and a puncture in the film could compromise the Miro Canvas. Larger pipette tips may not fully insert into the inlet, causing reagent regurgitation.

The detection method relies on both detection of a smart tag within the cartridge, and pressure exerted by a cartridge that has been securely clamped into place. A touchscreen Cartridge Missing Tag error may be resolved by ensuring the cartridge contains a smart tag. Touchscreen errors of Used or Expired Cartridge Detected may be resolved by ensuring a new, unexpired cartridge is being used. A touchscreen Cartridge Not Detected error may be resolved by opening the instrument lid, releasing the cartridge clamp, and firmly pressing down on the cartridge (without sliding it on the electrode board). Then, lower the cartridge clamp until the clip engages and close in the instrument lid by firmly pressing down on both corners. If this does not resolve the error, discard the current cartridge and load a new cartridge into the instrument.

To open the cartridge clamp, pull the clamp clip in the direction of the arrow until it disengages. To close the cartridge clamp, lower the clamp until the clip engages. Be sure to engage the clamp after loading the cartridge into the instrument, but before you begin loading reagents, as engaging the clamp too late may result in high voltage (HV) drop out. Also, ensure the cartridge clamp is engaged during sample recovery.

To open the instrument lid, secure the base of the instrument with one hand, grasp the front center of the lid with the other hand, and lift straight up. Do not tip the instrument, as this may move your sample away from the recovery inlet. To close the instrument lid, press down on both corners of the lid to engage both lid electromagnets. Keep the instrument lid closed whenever possible to prevent dust from contaminating the electrode board.

When your instrument is shipped to you, you will receive an email with instructions to set up an Organization for your group on the Miroculus web app. Protocols can be found on your Organization, and can be pushed from the Organization to your instrument. Both the computer used to access your Organization and the Miro Canvas instrument must be connected to the internet to be able to push protocols. Simply find the protocol of interest on your Organization and click the Send icon. Then choose the instrument you would like to send to from the dropdown menu and confirm. Protocols can be removed from your instrument by accessing the instrument touchscreen and tapping the Delete icon.

Miro Dropgloss has a freezing temperature of 18C. This means that if the temperature during a run gets too cold, the Dropgloss could solidify in the cartridge and cause the run to fail. As a result, if the temperature of the environment is between 16C – 18C, a warning will appear on screen to warn the user that the environment is too cold and should be warmed to avoid an error. If the temperature falls below 16C, the instrument will stop a run that is in progress. If the environment is below 16C before the run, it will not allow the run setup to begin.

An LED indicator bar of blue (solid) means the instrument is initializing, available, and ready.
An LED indicator bar of blue (dynamic gradient) means a protocol is running on the instrument.
An LED indicator bar of yellow means that a software error has occurred.
An LED indicator bar of a rainbow of colors means that a firmware error has occurred.
An LED indicator bar of green means that the instrument is performing a Miro Diagnostics check.

Level the instrument by lifting the lid and clamp, and placing the leveling plate on top of the electrode board. Twist the instrument feet left and right, one at a time, to raise and lower the instrument until the bubble is in the center of the leveling plate. If an instrument is not level, it can hinder mobility of droplets and cause a run to fail.

To connect the instrument to a network, ensure that the Wi-Fi antenna is securely screwed into the instrument, or that an Ethernet cord has been plugged in. However, do not attempt to connect to Wi-Fi while an Ethernet cord is plugged in. Use the touchscreen Settings menu to connect to a network. Protocols that are already loaded onto the instrument can be run without a connection to a network. However, it is best to remain connected to a network whenever possible. A network connection is required for updates to software and firmware, transferring protocols onto the instrument, and optimal technical support and troubleshooting.

After plugging the Miro Canvas to the power supply, power on your instrument by pressing the black power button on the back of the instrument. The instrument may take a few minutes to fully power on. Once fully booted, the touchscreen will depict a Start button and the LED indicator bar will turn blue. If the instrument does not successfully start up, power may not be flowing to the instrument. Ensure power supply cords are securely attached to the instrument, power brick and wall outlet, and that the wall outlet is supplying power.